JP2011020923A - Diagnosis and treatment of arteriosclerosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical composition which is effective for the treatment/prevention, detection or diagnosis of arteriosclerosis. <P>SOLUTION: In one aspect, there is provided a pharmaceutical composition for the diagnosis, treatment or prevention of a lesion of arteriosclerosis, which comprises a hydrophilized liposome and a factor specific to the lesion of arteriosclerosis. There is also provided a diagnostic composition for the diagnosis of a lesion of arteriosclerosis. The diagnostic composition comprises (A) the hydrophilized liposome, (B) the factor specific to the lesion of arteriosclerosis, and (C) a labeling substance. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a technique for preventing, detecting or diagnosing arteriosclerosis. The present invention relates to treating, preventing, detecting or diagnosing arteriosclerosis, including detecting specific factors for arteriosclerotic lesions, particularly vulnerable plaque. In particular, the present invention relates to treating, preventing, detecting or diagnosing a disease, disorder or condition associated with macrophages and treating, preventing, detecting or diagnosing arteriosclerosis.

  Deaths from cardiovascular diseases centered on arteriosclerotic diseases, myocardial infarction, and cerebrovascular disorders centered on cerebral infarction and stroke account for 30% of deaths. The frequency of aging ahead of our world is expected to increase more and more. It is very important to diagnose arteriosclerosis and start treatment immediately.

  Endothelial cells and macrophages (Mφ) play important roles in the formation of arteriosclerotic lesions. Unstable plaque is prone to failure. The stenosis or occlusion of the lumen associated with the failure causes cardiovascular disease and cerebrovascular disorder. If unstable plaque can be visualized, it is possible to start treatment before failure, which is very important medically (Patent Document 1: International Publication No. 2007/091661 and Non-Patent Document 1: Pathology of the). Vulnerable Plaque.Journal of the American College of Cardiology, Volume 47, 2006 Apr 18; 47 (8 Suppl): C13-8.Virmani, A. Burke, A. Farb, A. Farb.).

At present, the presence diagnosis of arteriosclerotic lesions is performed by a wide variety of methods. Although MRI, CT, and vascular endoscopy and intravascular echography are performed for quantitative and qualitative diagnosis of arteriosclerotic lesions, their accuracy is not sufficient. Therefore, an adhesion factor expressed in endothelial cells of arteriosclerotic lesions (Non-Patent Document 2: Nahrendorf et al., Circulation 2006; 114; 1504-1511 or Mφ secreted extracellular matrix degrading enzyme (Non-patent Document 3: Deguchi) et al., Circulation 114 (1): 55-62) have been experimentally attempted, but a method that can clearly diagnose unstable plaque has not been established at present.
International Publication No. 2007/091661 Pamphlet Pathology of the Vulnerable Plaque. Journal of the American College of Cardiology, Volume 47, 2006 Apr 18; 47 (8 Suppl): C13-8. Virmani, A .; Burke, A.M. Farb, F .; Kolodgie Nahrendorf et al. , Circulation 2006; 114; 1504-1511 Deguchi et al. , Circulation 114 (1): 55-62.

  An object of the present invention is to provide a method for qualitative diagnosis of arteriosclerotic lesions, and a technique for treating, preventing, detecting or diagnosing a disease, disorder or condition associated with macrophages.

  Another object of the present invention is to develop a technology capable of specifically reaching vulnerable plaque or macrophages and provide a new drug delivery system (DDS: drug delivery system) for treating, diagnosing or preventing arteriosclerosis It is in.

  The present inventors confirmed that fluorescently labeled liposomes were taken into the atherosclerotic lesion using in vivo imaging and a histological technique using an arteriosclerosis disease model mouse (FIGS. 1, 2 and 3). ). As a result, the liposome surface is modified with an antibody, sugar chain, or the like, so that it can be more specifically accumulated in the arteriosclerotic lesion and developed into an invention that increases the accuracy of imaging.

  The pharmaceutical composition of the present invention can diagnose, treat or prevent arteriosclerotic lesions by including hydrophilic liposomes and specific factors for arteriosclerotic lesions.

  The composition of the present invention is (1) specific for an arteriosclerotic lesion such as an antibody, an antibody fragment, an antibody variant, a sugar chain or a sugar chain group against an adhesion molecule specifically expressed in endothelial cells or plaques of the atherosclerotic lesion. Inclusion of a factor to increase accumulation in the sclerotic lesion; and (2) an antibody, antibody fragment, antibody variant, sugar chain or glycan group for a receptor specifically expressed in macrophages of the arteriosclerotic lesion By including a specific factor for macrophages such as, phagocytic efficiency by macrophages can be increased.

  The use of surface-modified liposomes as a diagnostic method for unstable plaques is a completely new method and an original method, and it can be said that detection efficiency and therapeutic efficiency that could not be achieved by the prior art can be achieved.

  In the composition of the present invention, a specific factor for arteriosclerotic lesions or macrophages (for example, an antibody, an antibody fragment, an antibody variant, a sugar chain against a molecule expressed on the endothelium of the atherosclerotic lesion or a receptor expressed on the macrophage surface) , A sugar chain group) is included in the hydrophilic liposome. Specific examples of antibodies include antibodies against cell adhesion molecules such as VCAM-1, ICAM-1, E-selectin, P-selectin or extracellular matrix degrading enzymes such as MMP, or antibody fragments or antibody variants thereof (for example, F (ab ′) 2 and Fab fragments, chimeric antibodies, and anti-idiotype antibodies and other recombinantly produced conjugates, etc.) can be used. As the sugar chain, sialyl Lewis X (SLX), SLX group, mannose, mannose group can be used.

  In the composition of the present invention, the specific factor for atherosclerotic lesions or macrophages may be encapsulated or bound to the hydrophilicized liposome.

  Hydrophilized liposomes used in the compositions of the present invention can be produced using the methods described herein.

  The composition of the present invention is used as an imaging agent for imaging arteriosclerotic lesions, macrophages or macrophage-related diseases by labeling with a labeling substance (fluorescent dye (eg, Cy3, Cy5.5), magnetic metal, etc.). Can also be used.

Accordingly, the present invention provides the following.
(1) A pharmaceutical composition for diagnosing, treating or preventing an arteriosclerotic lesion, comprising a hydrophilized liposome and a specific factor for arteriosclerotic lesions.
(2) The pharmaceutical composition according to the above item, wherein the specific factor for the arteriosclerotic lesion is selected from the group consisting of sialyl Lewis X (SLX), SLX group, mannose, mannose group, antibody, antibody fragment and antibody variant. object.
(3) The pharmaceutical composition according to the above item, wherein the specific factor for the arteriosclerotic lesion is sialyl Lewis X (SLX) or SLX group.
(4) The pharmaceutical composition according to the above item, wherein the diagnosis is a diagnosis of unstable plaque.
(5) The pharmaceutical composition according to the above item, wherein the hydrophilization is performed by a tris (hydroxymethyl) methylamino group.
(6) A part of the lipid constituting the liposome has a hydrophilic compound cross-linking group-W-tris (hydroxymethyl) methylamino group and NH-C (= O) bond, where W is The pharmaceutical composition according to the above item, which is a C (═O) —NH bond.
(7) The pharmaceutical composition according to the above item, wherein the liposome and the SLX group are bound via a human serum albumin group.
(8) A part of the lipid constituting the liposome is bonded to human serum albumin group-A-linker protein cross-linking group-X-SLX group and CH ₂ —NH, where A is NH—C The pharmaceutical composition according to the above item, which is a (═O) bond, and X is a C (═O) —NH bond.
(9) The pharmaceutical composition according to the above item, wherein the SLX group is present on the surface of the liposome.
(10) A diagnostic composition for diagnosing arteriosclerotic lesions, comprising:
A) a hydrophilized liposome;
B) specific factors for arteriosclerotic lesions;
C) A diagnostic composition comprising a labeling substance.
(11) The diagnostic agent according to the above item, wherein the specific factor for the arteriosclerotic lesion is selected from the group consisting of sialyl Lewis X (SLX), SLX group, mannose, mannose group, antibody, antibody fragment and antibody variant Composition.
(12) The diagnostic composition according to the above item, wherein the specific factor for the atherosclerotic lesion is sialyl Lewis X (SLX) or SLX group.
(13) The diagnostic composition as described in the above item, for use in magnetic resonance imaging.
(14) The diagnostic composition according to the above item, wherein the diagnosis is a diagnosis of unstable plaque.
(15) The diagnostic composition according to the above item, wherein the hydrophilization is performed by a tris (hydroxymethyl) methylamino group.
(16) A part of the lipid constituting the liposome has a hydrophilic compound cross-linking group -W-tris (hydroxymethyl) methylamino group and NH-C (= O) bond, where W is The diagnostic composition according to the above item, which is a C (═O) —NH bond.
(17) The diagnostic composition according to the above item, wherein the liposome and the SLX group are bound via a human serum albumin group.
(18) A part of the lipid constituting the liposome is bonded to human serum albumin group-A-linker protein cross-linking group-X-SLX group and CH ₂ —NH, where A is NH—C The diagnostic composition according to the above item, which is a (═O) bond, and X is a C (═O) —NH bond.
(19) The diagnostic composition according to the above item, wherein the SLX group is present on the surface of the liposome.
(20) The diagnostic composition according to the above item for diagnosing an arteriosclerotic lesion by magnetic resonance imaging, wherein the labeling substance is a substance that can be detected by nuclear magnetic resonance imaging Composition.
(21) The diagnostic composition according to the above item, wherein the labeling substance is a paramagnetic metal.
(22) A method for treating or preventing arteriosclerotic lesions, which comprises administering an effective amount of the pharmaceutical composition as described above to a patient in need of treating or preventing atherosclerotic lesions A method comprising the steps.
(23) A method for detecting an arteriosclerotic lesion, the method comprising:
Administering an effective amount of the diagnostic composition described in the above item to a patient in need of detecting an arteriosclerotic lesion; and, in the patient, labeling substance contained in the diagnostic composition is detected by magnetic resonance imaging. A method comprising measuring and indicating that the presence of the labeling substance indicates an arteriosclerotic lesion in the patient.
(24) Use of a compound containing a specific factor for arteriosclerotic lesions in the manufacture of a medicament for treating, preventing, detecting or diagnosing arteriosclerotic lesions.
(25) A macrophage or a macrophage-related disease, disorder or disease comprising a hydrophilized liposome and a specific factor for macrophage selected from the group consisting of mannose, mannose group, antibody, antibody fragment and antibody variant A pharmaceutical composition for diagnosing, treating or preventing a condition.
(26) The pharmaceutical composition according to the above item, wherein the diagnosis, treatment or prevention includes targeting only atherosclerotic lesions in which highly active macrophages are present.
(27) A diagnostic composition for diagnosing a macrophage or a disease, disorder or condition associated with macrophage, comprising the following:
A) a hydrophilized liposome;
B) a specific factor for macrophages selected from the group consisting of mannose, mannose groups, antibodies, antibody fragments and antibody variants;
C) A diagnostic composition comprising a labeling substance.
(28) The diagnostic composition according to the above item, wherein the diagnosis includes diagnosing only an arteriosclerotic lesion in which highly active macrophages are present.

  Accordingly, these and other advantages of the invention will be apparent upon reading the following detailed description.

According to the present invention, it has become possible to image unstable plaque, which has been impossible or insufficient in the past, and not only the existence diagnosis of arteriosclerotic lesions but also qualitative diagnosis.
New inspection methods can be developed.

  The present invention will be described below with reference to the best mode. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the above field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

  Hereinafter, definitions of terms particularly used in the present specification will be described as appropriate.

  In this specification, “arteriosclerosis” refers to a phenomenon in which an artery becomes hard. Examples thereof include a state in which the elasticity and flexibility have been lost due to thickening of the arterial wall and lipid deposition including aortic atherosclerosis and arteriole sclerosis.

  In the present specification, the “arteriosclerotic lesion” refers to a portion of a lesion site where atherosclerosis is observed, particularly a portion forming a plaque.

  As used herein, “specific factor for arteriosclerotic lesion” refers to a factor that is specifically expressed in arteriosclerotic lesion. These include, for example, sialyl Lewis X (SLX), SLX group, mannose, mannose group, antibodies (for example, antibodies against cell adhesion molecules such as VCAM-1, ICAM-1, E-selectin, P-selectin, MMP, etc. Antibodies against extracellular matrix degrading enzymes, etc.), antibody fragments, antibody variants and the like.

  In the present specification, the “compound containing a specific factor for arteriosclerotic lesion” refers to a compound containing a factor specific for arteriosclerotic lesion.

  As used herein, “arteriosclerotic lesion” refers to a lesion in which atherosclerosis is observed, for example.

  In this specification, “diagnosis” of an arteriosclerotic lesion refers to, for example, determining an atherosclerotic lesion by some means.

  In the present specification, “treating” an arteriosclerotic lesion refers to, for example, once an atherosclerotic lesion has occurred, treating the disorder by some means to cure, improve, prevent deterioration, and the like.

  As used herein, “preventing” an arteriosclerotic lesion means that, for example, before the atherosclerotic lesion occurs, the lesion is not caused or at least delayed, or the condition itself has occurred. Even so, it should be in a state that does not cause a failure.

  In this specification, “detecting” an arteriosclerotic lesion refers to, for example, determining the presence or absence of an atherosclerotic lesion, and in particular, visually identifying the presence or absence of an atherosclerotic lesion from the outside of the body. To do.

  In the present specification, “unstable plaque” means that inflammatory cells such as macrophages are actively infiltrated among atherosclerotic lesions, and the inside is rich in lipids, and the coating covering the surface is very thin, A lesion that can easily break down.

  In this specification, “diagnosis of unstable plaque” refers to determining the presence or absence of unstable plaque by some means.

  As used herein, “macrophages” refer to mononuclear and active phagocytes.

  As used herein, “disease associated with macrophages” refers to any disease associated with the state of macrophages, for example, diseases associated with inflammatory diseases including atherosclerotic lesions.

  As used herein, “a disorder associated with macrophages” refers to any disorder associated with the state of macrophages, such as disorders associated with inflammatory diseases including atherosclerotic lesions.

  As used herein, “a state associated with macrophages” refers to any state of macrophages, such as a state associated with inflammatory diseases including atherosclerotic lesions.

  In this specification, “diagnosis” of a disease associated with macrophages refers to determining by some means whether or not a disease associated with macrophages has occurred.

  “Treatment” of a disease associated with macrophages refers to the treatment of the disorder by some means after the disease associated with macrophages has occurred to cure, improve, prevent deterioration, and the like.

  “Preventing” a macrophage-related disease means that before the macrophage-related disease develops, by any means it does not cause or at least delays the disease, or even if the condition itself occurs. It means that the cause is not damaged.

  “Diagnosing” a disorder associated with macrophages means determining by some means whether or not a disorder associated with macrophages has occurred.

  “Treatment” of a macrophage-related disorder refers to treatment of the disorder by some means once the macrophage-related disorder has occurred to cure, improve, prevent deterioration, and the like.

  “Preventing” a macrophage-related disorder means that before a macrophage-related disorder occurs, it is not caused, or at least delayed, by any means, even if the disorder itself occurs. It means that the cause is not damaged.

  “Diagnosing” a condition associated with macrophages means determining by some means whether or not a condition associated with macrophages has occurred.

  “Treatment” of a condition associated with macrophages refers to treatment of the disorder by some means after the condition associated with macrophages has occurred, to cure, improve, prevent deterioration, and the like.

  “Preventing” a condition associated with a macrophage means that the condition associated with the macrophage does not occur or is at least delayed by any means before the condition associated with the macrophage occurs, or if the condition itself occurs. It means that the cause is not damaged.

  As used herein, “highly active macrophages” refers to macrophages that are activated by inflammatory cytokines to produce MMP and actively incorporate oxidized LDL. Such an activation level can be determined by a level considered to be active in the field using a technique known in the field.

  As used herein, “statin” refers to a substance that specifically and antagonistically inhibits HMG-CoA reductase, which is the rate-limiting enzyme of the cholesterol biosynthesis system, and is also referred to as an HMG-CoA reductase inhibitor. Say. Examples of these include, but are not limited to, pravastatin, atorvastatin, simvastatin, lovastatin, fluvastatin, and vitavastatin.

  In the present specification, the “sugar chain” refers to a compound formed by connecting one or more unit sugars (monosaccharide and / or a derivative thereof). When two or more unit sugars are connected, each unit sugar is bound by dehydration condensation by a glycosidic bond. Examples of such sugar chains include polysaccharides (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and complexes and derivatives thereof) contained in the living body. Other examples include, but are not limited to, a wide range of sugar chains that are decomposed or derived from complex biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, glycolipids, and the like. Therefore, in the present specification, the sugar chain can be used interchangeably with “polysaccharide”, “carbohydrate”, and “carbohydrate”. Further, unless otherwise specified, the “sugar chain” in the present specification may include both sugar chains and sugar chain-containing substances. Typically, a substance in which about 20 types of monosaccharides (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and complexes and derivatives thereof) are connected in a chain. It is attached to proteins and lipids inside and outside the cells of the body. The functions differ depending on the sequence of monosaccharides, and they are usually branched in a complex manner. The human body is expected to have several hundreds of sugar chains with various structures, and there are tens of thousands of useful structures in the human body. It is believed that there are more than types. Although it is considered to be related to higher-order functions that proteins and lipids perform in vivo, such as cell-to-cell molecular and cell recognition functions, there are many unexplained mechanisms. It is attracting attention in current life science as the third life chain after nucleic acid and protein. In particular, the function of a sugar chain as a ligand (information molecule) in cell recognition is expected, and its application to the development of highly functional materials is being studied.

  In this specification, the “sugar chain group” is a name given when a sugar chain is bonded to another group. The sugar chain group refers to a monovalent or divalent group depending on the case. For example, examples of the sugar chain group include a sialyl Lewis X group, an N-acetyllactosamine group, and an α1-6 mannobiose group.

  As used herein, “sugar” or “monosaccharide” refers to polyhydroxyaldehyde or polyhydroxyketone containing at least one hydroxyl group and at least one aldehyde group or ketone group, and constitutes the basic unit of a sugar chain. In this specification, sugar is also referred to as carbohydrate and both are used interchangeably. In this specification, when specifically mentioned, a sugar chain refers to a chain or a sequence in which one or more sugars are linked, and when referred to as a sugar or a monosaccharide, it refers to one unit constituting the sugar chain. Here, those in which n = 2, 3, 4, 5, 6, 7, 8, 9 and 10 are referred to as diose, triose, tetrose, pentose, hexose, heptose, octose, nonose and decourse, respectively. Generally, it corresponds to an aldehyde or ketone of a chain polyhydric alcohol. The former is called aldose and the latter is called ketose. In the present invention, any type can be used.

  The nomenclature and abbreviations used to describe sugars in the present invention follow conventional nomenclature. For example, β-D-galactose

Is Gal;
N-acetyl-α-D-galactosamine

Is GalNAc;
α-D-mannose

Is Man;
β-D-glucose

Is Glc;
N-acetyl-β-D-glucosamine

Is GlcNAc;
α-L-Fucose

Is Fuc;
α-N-acetylneuraminic acid

Is Neu5Ac;
Ceramide

Is Cer;
L- serine _{CH 2 (OH) CH (COOH} ) NH 2
Is represented by Ser. Cer is usually classified as a lipid, but in the present specification, it is also treated as a sugar unless otherwise specified because it also falls within the definition of a kind of sugar constituting a sugar chain. In addition, Ser is usually classified as an amino acid, but in the present specification, it is also treated as a saccharide unless otherwise mentioned because it also falls within the definition of a kind of saccharide constituting a glycan. Two cyclic anomers are represented by α and β. For reasons of display, it may be expressed as a or b. Therefore, in the present specification, α and a, and β and b are used interchangeably for the anomeric notation.

  In this specification, galactose refers to any isomer, but is typically β-D-galactose, and is used to refer to β-D-galactose unless otherwise specified.

  In this specification, acetylgalactosamine refers to any isomer, but is typically N-acetyl-α-D-galactosamine, and refers to N-acetyl-α-D-galactosamine unless otherwise specified. Used as a thing.

  In this specification, mannose refers to any isomer, but is typically α-D-mannose, and is used to refer to α-D-mannose unless otherwise specified.

  In this specification, glucose refers to any isomer, but is typically β-D-glucose, and is used to refer to β-D-glucose unless otherwise specified.

  In this specification, acetylglucosamine refers to any isomer, but is typically N-acetyl-β-D-glucosamine, and unless otherwise specified, refers to N-acetyl-β-D-glucosamine. Used as a thing.

  In this specification, fucose refers to any isomer, but is typically α-L-fucose, and is used to refer to α-L-fucose unless otherwise specified.

  In this specification, acetylneuraminic acid refers to any isomer, but is typically α-N-acetylneuraminic acid, and unless otherwise specified refers to α-N-acetylneuraminic acid. Used as.

  In the present specification, serine refers to any isomer, but is typically L-serine, and is used to refer to L-serine unless otherwise specified.

  In this specification, it is noted that the symbol, designation, abbreviation (Glc, etc.), etc. of sugars are different when they represent monosaccharides and when used in sugar chains. In the sugar chain, the unit sugar exists in a form in which hydrogen or a hydroxyl group is removed from the other side because dehydration condensation occurs between the unit sugar and another unit sugar to be bound. Therefore, when these sugar abbreviations are used to represent monosaccharides, all hydroxyl groups are present, but when used in a sugar chain, the hydroxyl groups of the sugars to which the hydroxyl groups are bound are dehydrated. It is understood that only oxygen remains after condensation.

  When a sugar is covalently bonded to albumin, the reducing end of the sugar is aminated and can be bound to other components such as albumin via its amine group, in which case the hydroxyl group at the reducing end is an amine group. Note that it has been replaced with.

  Monosaccharides are generally joined by glycosidic bonds to form disaccharides and polysaccharides. The bond orientation with respect to the plane of the ring is indicated by α and β. Specific carbon atoms that form a bond between two carbons are also described.

  In this specification, the sugar chain is

Is represented by Thus, for example, the β glycosidic bond between C-1 of galactose and C-4 of glucose is represented by Galβ1,4Glc. Thus, for example, sialyl Lewis X (SLX) is represented as Neu5Acα2,3Galβ1,4 (Fucα1,3) GlcNAc. N-acetyllactosamine (G4GN) is represented as Galβ1,4GlcNAc. α1-6 mannobiose (A6) is expressed as Manα1,6Man.

  A sugar chain branch is represented by parentheses and is placed immediately to the left of the unit sugar to be bound. For example,

And in parentheses are

Is written. Thus, for example, when β-glycosidic bond is formed between C-1 of galactose and C-4 of glucose, and C-3 of glucose is further α-glycoside bonded to C-1 of fucose, Galβ1,4 ( It is expressed as Fucα1,3) Glc. Monosaccharides are represented on the basis of numbering as low as possible to (latent) carbonyl groups. Even when an atomic group superior to a (latent) carbonyl group is introduced into a molecule according to the general standard of organic chemical nomenclature, it is usually represented by the above numbering.

  Examples of the sugar chain used in the present specification include sugar chains selected from the group consisting of sialyl Lewis X, N-acetyllactosamine, α1-6 mannobiose, and combinations of two or more thereof. It is not limited to. The reason why two or more combinations can be used is not limited by theory, but each of the sugar chains has specificity for a lectin localized in the tissue or cell of the target delivery site, and is mixed. This is because it is considered that the specificity is exhibited.

  As used herein, the term “antibody” refers to an immunoglobulin molecule having a specific amino acid sequence elicited by an antigen that is an immunogen. Antibodies are produced by B cells and are present in blood and body fluids. An antibody has the characteristic of reacting specifically with an antigen. The antibody may be present naturally rather than as a result of stimulation by presentation of the antigen. Basically, the molecular structure of an antibody is formed by two light chains and a heavy chain, but can also exist as a dimer, trimer, or pentamer. Examples of these include, but are not limited to, IgA, IgE, IgM, IgG, and the like. Examples of the antibody used in the pharmaceutical composition of the present invention include an antibody against an antigen specifically expressed in an organ, a disease, an immune cell and the like. Preferably, for example, antibodies against cell adhesion molecules such as VCAM-1, ICAM-1, E-selectin and P-selectin expressed on inflammatory vascular endothelium, and antibodies against extracellular matrix degrading enzymes such as MMP, It is not limited to. In one embodiment, the antibody that can be used in the pharmaceutical composition of the invention can be, for example, an anti-EGFR antibody, IgG.

  In the present specification, the “antibody variant” refers to one in which a part of the original antibody is changed. In the present specification, the “antibody” includes a polyclonal antibody, a monoclonal antibody, a multispecific antibody, a chimeric antibody, an anti-idiotype antibody, and the like. As the antibody “variant”, for example, an antibody fragment such as F ( ab ') 2 and Fab fragments, as well as other recombinantly produced conjugates. In addition, such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, and the like. In the present specification, “antibody fragment” or “antibody fragment” refers to a polypeptide having an amino acid sequence length of 1 to n−1 with respect to a full-length antibody (length is n). The length of the fragment can be appropriately changed according to the purpose, and the lower limit of the length may be short as long as it functions as an antibody. For example, 3, 4, 5, 6, 7, 8 , 9, 10, 15, 20, 25, 30, 40, 50 and more, and the lengths represented by integers not specifically listed here (eg, 11 etc.) are also It may be appropriate as a lower limit. In the present specification, the length of a polypeptide can be represented by the number of amino acids as described above. However, the above number is not absolute, and as long as it has the same function, the above-described upper limit or lower limit. The number of is intended to include those that are several above and below that number (or, for example, 10% above and below). In order to express such intention, in this specification, “about” may be added before the number.

  In the present specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value. The length of a fragment useful herein can be determined by whether or not at least one of the functions of a full-length antibody that serves as a reference for the fragment is retained.

(Liposome)
In the present specification, the “liposome” usually means a closed vesicle composed of a lipid layer assembled in a membrane shape and an inner aqueous layer. In addition to phospholipids typically used, cholesterol, glycolipids and the like can also be incorporated. Since liposomes are closed vesicles containing water inside, it is possible to retain water-soluble drugs and the like in the vesicles. Therefore, such liposomes are used to deliver drugs and genes that cannot pass through the cell membrane into the cell. Moreover, since biocompatibility is good, the expectation as a nanoparticulate carrier material for DDS is great. In the present invention, the liposome is a structural unit having a functional group that imparts an ester bond (eg, glycolipid, ganglioside, phosphatidylglycerol, etc.) or a structural unit that has a functional group that imparts a peptide bond in order to attach a modifying group. (Eg, phosphatidylethanolamine).

  The liposome can be prepared by any technique known in the art. For example, among them, a method using a cholic acid dialysis method is exemplified. In the cholate dialysis method, production is carried out by a) preparation of mixed micelles of lipid and surfactant, and b) dialysis of mixed micelles. The liposome used in the composition of the present invention may contain a sugar chain, a sugar chain group, an antibody, an antibody fragment, and an antibody variant. Liposomes in which sugar chains, antibodies, and the like are bound to liposomes can also be used in the pharmaceutical composition of the present invention. In this case, it is preferable to use a protein as a linker. For example, coupling of a glycoprotein in which a sugar chain is bound to a protein to a liposome can be performed by the following two-step reaction. a) periodate oxidation of the ganglioside moiety on the liposome membrane, and b) coupling of the glycoprotein to the oxidized liposome by reductive amination reaction. By such a technique, a glycoprotein containing a desired sugar chain can be bound to the liposome, and a wide variety of glycoprotein / liposome conjugates having the desired sugar chain can be obtained. It is very important to examine the particle size distribution to see the purity and stability of the liposomes. As the method, gel filtration chromatography (GPC), scanning electron microscope (SEM), dynamic light scattering (DLS), or the like can be used. Dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate can be produced in the type 35: 40: 15: 5: 5: 167 type liposome. The liposome is stable even when stored at 4 ° C. for several months. The in vivo stability of liposomes can be examined using mice. Liposomes are intravenously injected into mice, blood is collected after 3 hours to prepare serum, and ultrafiltration is performed using a membrane having a pore size of 0.03 μm to purify and collect the liposomes. As a result of the SEM observation, it can be confirmed that the form of the liposome is not changed before and after the treatment and recovery for 3 hours in vivo.

  As used herein, the term “lipid” refers to a long chain aliphatic hydrocarbon or derivative thereof. “Lipid” is a general term for compounds composed of, for example, fatty acids, alcohols, amines, aminoalcohols, aldehydes and the like. Examples of the lipid constituting the liposome used in the present invention include phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, long-chain alkyl phosphates, glycolipids (gangliosides, etc.), phosphatidylglycerols, sphingomyelins, and the like. And cholesterols.

  Examples of the phosphatidylcholines include dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, and the like.

  Examples of the phosphatidylethanolamines include dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, and the like.

  Examples of the phosphatidic acids include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, and distearoyl phosphatidic acid. Examples of long-chain alkyl phosphates include dicetyl phosphate.

  Examples of glycolipids include galactosylceramide, glucosylceramide, lactosylceramide, phosphnatide, globoside, gangliosides and the like. Examples of gangliosides include ganglioside GM1 (Galβ1,3GalNAcβ1,4 (NeuAα2,3) Galβ1,4Glcβ1,1′Cer), ganglioside GDla, ganglioside GTlb, and the like.

  As the phosphatidylglycerols, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol and the like are preferable.

  Of these, phosphatidic acids, long-chain alkyl phosphates, glycolipids, and cholesterols have the effect of increasing the stability of liposomes, and are therefore preferably added as constituent lipids. For example, as the lipid constituting the liposome used in the present invention, phosphatidylcholines (molar ratio 0 to 70%), phosphatidylethanolamines (molar ratio 0 to 30%), phosphatidic acids, and long-chain alkyl phosphates are used. One or more lipids selected from the group consisting of (1 to 30% molar ratio), one or more lipids selected from the group consisting of glycolipids, phosphatidylglycerols and sphingomyelin (molar ratio 0 to 40%) ), And cholesterol (molar ratio 0 to 70%). Preferably it contains gangliosides, glycolipids or phosphatidylglycerols. This is because a linker such as albumin is easily bonded.

  In a preferred embodiment, the liposome used in the pharmaceutical composition of the present invention contains ganglioside, glycolipid or phosphatidylglycerol, to which a linker such as a peptide is bound, and to which a sugar chain, an antibody or the like can be bound. is there.

  By combining ganglioside, glycolipid, or phosphatidylglycerol when preparing the liposome, it is possible to prepare a liposome containing a sugar chain contained in the glycolipid as a constituent component.

  In another preferred embodiment, the liposome used in the pharmaceutical composition of the present invention preferably comprises phosphatidylethanolamine. This is because inclusion of phosphatidylethanolamine facilitates bonding with a hydrophilicity-imparting group (such as tris (hydroxyalkyl) aminoalkane).

  In the present specification, the “sugar chain-modified liposome” or “liposome modified with a sugar chain” refers to a substance containing a sugar chain and a liposome, preferably modified by direct or indirect binding of sugar chains. Refers to liposomes.

  As used herein, “antibody-modified liposome” or “antibody-modified liposome” refers to a substance containing an antibody, an antibody fragment or an antibody variant (sometimes referred to herein as “antibody or the like”) and a liposome. Preferably, it refers to a liposome modified by direct or indirect binding of an antibody or the like.

Specifically expressing the form in which sugar chains or antibodies are bound to liposomes,
Structure I X-R ¹ -R ² -R ³
X: a group in which the functional group a is removed from a structural unit containing a functional group a capable of bonding with the linker protein and CH ₂ —NH in the liposome R ¹ : linker protein group R ² : linker protein cross-linking group R ³ : A sugar chain or an antibody, wherein X and R ¹ are CH ₂ —NH bonded, R ¹ and R ² are peptide bonded, and R ² and R ³ are peptide bonded.
Thus, in more detail, structure I is
X—CH ₂ —NH—R ¹ —NH—C (═O) —R ² —C (═O) —NH—R ³
It can be represented by the structural formula

Liposomes used in the present invention are rendered hydrophilic by the following structure:
Structure II YR ⁴ -R ⁵
Y: a group in which the functional group b is removed from a structural unit containing a hydrophilic compound crosslinking group and a peptide-bondable functional group b contained in the liposome R ⁴ : a hydrophilic compound crosslinking group R ⁵ : a hydrophilic compound group Y R ⁴ is a peptide bond, and R ⁴ and R ⁵ are peptide bonds.
Thus, in more detail, structure II is
Y—NH—C (═O) —R ⁴ —C (═O) —NH—R ⁵
It can be represented by the structural formula

  In the pharmaceutical composition of the present invention, liposomes having the above-mentioned structure I and structure II and further fluorescent can be used.

  In the present invention, the fluorescence is imparted by at least one of the components of the liposome having fluorescence, or when the liposome further has an element having fluorescence (for example, a fluorescent dye).

  Examples of the fluorescent element include, but are not limited to, a fluorescent dye, a fluorescent protein (eg, GFP, CFP, YFP, etc.), and a luminescent enzyme (eg, luciferase). As the fluorescent dye, for example, cy5.5 (for example,

1,1′-bis (ε-carboxypentyl) -3,3,3 ′, 3′-tetramethyl-3H-benzindodicarbocyanine-5,5 ′, 7,7′-tetrasulfonate tripotassium salt -N-hydroxysuccinimide ester, etc.)
cy3 (for example,

1- (ε-carboxypentyl) -1′-ethyl-3,3,3 ′, 3′-tetramethylindocarbocyanine-5,5′-disulfonate potassium salt—N-hydroxysuccinimide ester (cy3), etc. ), Cy5, cy7, cy3B, cy3.5, Alexa
Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa
Fluor 647, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, fluorescein-4-isothiocyanate (FITC) and a fluorescent dye selected from the group consisting of europium-containing labels and combinations thereof. This is because the labeling substance only needs to make the composition detectable.

  The liposome used in the present invention is newly labeled with a paramagnetic element to give a label. For example, paramagnetism is a paramagnetic metal (eg, gadolinium, iron, etc.), a contrast agent (eg, iron oxide particles, gadolinium chelator, barium sulfate, water-soluble iodine, iohexol, iopamidol, urografin, etc.), ultrasound It can be applied by a contrast agent (for example, levovist, sonazoid, etc.), but is not limited thereto. This is because the labeling substance only needs to make the composition detectable in magnetic resonance imaging.

  As used herein, “compatible” with a liposome means that the substance does not impair the stability of the liposome when it is included in or bound to the liposome. Refers to nature. Compatibility with liposomes can be determined, for example, by measuring zeta potential, electric mobility, particle diameter, lipid amount, protein amount, and the like. For example, the aggregation property of the liposome particles can be determined by the zeta potential. The average particle size, the maximum region, the number of liposomes in the maximum region, and the like are analyzed based on the particle size and particle size distribution, and the uniformity of the liposomes can be confirmed based on the analysis result. By measuring the ratio of the amount of protein: the amount of lipid: the amount of protein per lipid, it can be confirmed whether the liposome has an appropriate composition.

  The sugar chain-modified liposomes and antibody-modified liposomes used herein can be included at a density suitable for delivery to the intended delivery site.

  As used herein, “modified bond density” is the amount of sugar chain used when preparing a sugar chain-modified liposome, and the density of sugar chains bound per mg of liposome lipid (mg sugar). Chain / mg lipid). The binding density of this sugar chain-modified liposome is not desired to be bound by theory, but empirically, the amount of sugar chain used in the preparation is approximately proportional to the density of the sugar chain bound to the liposome. I know that Therefore, in this specification, unless otherwise stated, the bond density is determined by the amount used at the time of preparation. In vitro, for example, it can be determined indirectly using E-selectin. The sugar chain-modified liposome used in the present invention can control the directivity with respect to the intended delivery site by selecting the type and the binding density of the sugar chain bound to the liposome. Table 1 below shows liposome numbers, sugar chain structures, and modified bond densities.

  As described in the above table, preferred sugar chain-modified liposomes in the present invention can be produced by the following method. Specifically, this method comprises the steps of: (a) suspending a lipid in a methanol / chloroform solution and stirring; evaporating the stirred solution; and drying the precipitate in vacuum to obtain a lipid membrane; (B) suspending the lipid membrane in a suspension buffer and sonicating; (c) mixing the sonicated solution with a fluorescent labeling solution to provide fluorescently labeled liposomes; d) hydrophilizing the liposome with tris (hydroxyalkyl) aminoalkane; (e) binding a linker protein to the hydrophilized liposome to produce a linker protein-bound liposome; and f) A step of producing a sugar chain-modified liposome by binding the sugar chain described in the above table to the liposome is included.

  Preferably, in this method, the fluorescent labeling solution of step (c) is 1,1′-bis (ε-carboxypentyl) -3,3,3 ′, 3′-tetramethylindocarbocyanine-5,5 ′. -Disulfonate potassium salt, di-N-hydroxysuccinimide ester (cy5.5), the linker protein in step (e) is human serum albumin, and in step (f), the sugar chain and the liposome Glycosylated liposomes are produced by conjugation under conditions suitable for delivery to the delivery site.

  The liposome and the linker, and the linker and the sugar chain are preferably bonded using a bifunctional crosslinking group (for example, 3,3′-dithiobis (sulfosuccinimidyl propionate) (DTSSP)). .

The antibody-modified liposome used in the present invention can be prepared by the same method as the sugar chain-modified liposome. That is, such an antibody-modified liposome can be produced by the following method. Specifically, the method comprises: A) providing a liposome; B) contacting the liposome with serum albumin under conditions under which the liposome and serum albumin are bound to produce a serum albumin-bound liposome. And C) a step of contacting the serum albumin-binding liposome and the antibody in the presence of a spacer under the condition that the serum albumin and the antibody are bound via the spacer to produce an antibody-modified liposome. Is included.

  Liposomes used in the present invention can encapsulate or bind drugs or genes. Examples of the drug include alkylated anticancer agents, antimetabolites, plant-derived anticancer agents, anticancer antibiotics, biological response modifiers (BRM) / cytokines, platinum complex anticancer agents, immunotherapeutic agents , Hormone anticancer agents, tumor drugs such as monoclonal antibodies, central nervous system drugs, peripheral nervous system / sensory organ drugs, respiratory disease drugs, cardiovascular drugs, gastrointestinal drugs, hormone drugs, urology / genital organs Drugs, vitamins / nutrient tonics, metabolic drugs, antibiotics / chemotherapeutic drugs, testing drugs, anti-inflammatory drugs, eye disease drugs, central nervous system drugs, autoimmune drugs, cardiovascular drugs, diabetes, high Lifestyle-related diseases such as lipemia, corticosteroids, immunosuppressive agents, antibacterial agents, antiviral agents, angiogenesis inhibitors, cytokines , Chemokines, anti-cytokine antibodies, anti-chemokine antibodies, anti-cytokine / chemokine receptor antibodies, siRNA, miRNA, smRNA, antisense oligodeoxynucleotide (ODN) or DNA-related nucleic acid preparations, neuroprotective factors Antibody drugs, molecular targeted drugs, osteoporosis / bone metabolism improving drugs, neuropeptides, bioactive peptides / proteins, but are not limited thereto.

  As used herein, a “linker” is a molecule that mediates the binding between a sugar chain, an antibody, and the like and the liposome surface. In the sugar chain-modified liposome or antibody-modified liposome used in the present invention, the sugar chain or antibody may be bound to the liposome surface via a linker. The linker can be appropriately selected by those skilled in the art, but is preferably biocompatible, and more preferably pharmaceutically acceptable. As used herein, “linker protein” refers to a protein, peptide, or amino acid polymer among linker molecules. The linker protein used in the present specification can be, for example, a biological protein, preferably a human-derived protein, more preferably a human-derived serum protein, and even more preferably serum albumin. In particular, when human serum albumin is used, it has been confirmed by experiments on mice that the uptake into each tissue is large.

  In the present specification, the “linker (protein) group” is a name given when a linker (protein) is bonded to another group. A linker (protein) group refers to a monovalent or divalent group depending on the case. Examples thereof include a mammal-derived protein group, a human-derived protein group, a human serum protein group, and a serum albumin group. The linker (protein) group is preferably derived from “human”. This is because the compatibility with human administration is considered high. Also preferred are proteins that are not immunogenic.

  In the present specification, the “crosslinking group” refers to a group that forms a chemical bond between molecules of a chain polymer so as to form a bridge. Typically, it acts between macromolecules such as lipids, proteins, peptides, and sugar chains and other molecules (eg, lipids, proteins, peptides, sugar chains), and is covalently bonded within or between molecules. It refers to a group that forms a covalent bond that connects places that did not exist. In the present specification, the crosslinking group varies depending on the target for crosslinking, and examples thereof include, but are not limited to, aldehydes (for example, glutaraldehyde), carbodiimides, and imide esters. When the amino group-containing substance is crosslinked, an aldehyde-containing group such as glutaraldehyde can be used.

  In the present specification, the “linker protein cross-linking group” refers to a group that forms a peptide bond between a liposome and a sugar chain. The linker protein cross-linking group varies depending on the target for cross-linking, such as bissulfosuccinimidyl suberate, disuccinimidyl glutarate, dithiobis succinimidyl propionate, disuccinimidyl suberate, 3 , 3′-dithiobis (sulfosuccinimidyl propionate), ethylene glycol bissuccinimidyl succinate, ethylene glycol bissulfosuccinimidyl succinate, and the like can be used. Examples of linker protein crosslinking groups that can be used in the present specification include 3,3′-dithiobis (sulfosuccinimidylpropionate) group, bissulfosuccinimidyl suberate group, and disuccinimidyl glutarate. Groups, dithiobissuccinimidyl propionate groups, disuccinimidyl suberate groups, ethylene glycol bissuccinimidyl succinate groups and ethylene glycol bissulfosuccinimidyl succinate groups.

  As used herein, the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also encompass one assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptide-like compounds (eg, peptoids) and other modifications known in the art. Is included.

  In this specification, “protein” refers to a polymer of an amino acid having a relatively large molecular weight or a variant thereof, and “peptide” refers to a polymer of an amino acid having a relatively small molecular weight or a modification thereof. It should be understood that it may refer to a variant. Examples of such molecular weight include, but are not limited to, about 30 kDa, preferably about 20 kDa, more preferably about 10 kDa.

  As used herein, “biological protein” refers to a protein derived from an organism, and any organism (eg, any type of multicellular organism (eg, an animal (eg, a vertebrate, an invertebrate)). Animal), plant (for example, monocotyledonous plant, dicotyledonous plant, etc.))) or the like. Preferably, it is a protein derived from a vertebrate (for example, larvae, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.), more preferably a mammal (for example, single pores, marsupials) , Rodents, crustaceans, wings, carnivores, carnivores, long noses, odd hoofs, even hoofs, rodents, scales, sea cattle, cetaceans, primates, rodents Or the like from the order of rabbits). More preferably, a protein derived from a primate (eg, chimpanzee, Japanese monkey, human) is used. Most preferably, a protein derived from a living body intended for administration is used. In this specification, when a biological protein shows the state couple | bonded with another substance, it is called a biological protein group.

  As used herein, “human-derived serum protein” refers to a protein contained in a liquid portion that remains when human blood naturally coagulates. In this specification, when a human-derived protein shows a state of binding to another substance, it is referred to as a human-derived protein group.

  As used herein, “serum albumin” refers to albumin contained in serum. In this specification, when serum albumin shows the state couple | bonded with another substance, it is called a serum albumin group.

  In the present invention, in the sugar chain-modified liposome and the antibody-modified liposome, at least one of the liposome membrane or the linker may be rendered hydrophilic by binding a hydrophilic compound, preferably a tris (hydroxyalkyl) aminoalkane.

  As used herein, “hydrophilization” refers to binding of a hydrophilic compound to the liposome surface. Examples of the compound used for hydrophilization include a low molecular weight hydrophilic compound, preferably a low molecular weight hydrophilic compound having at least one OH group, and more preferably a low molecular weight hydrophilic compound having at least two OH groups. Can be mentioned. Further, a low molecular weight hydrophilic compound having at least one amino group, that is, a hydrophilic compound having at least one OH group and at least one amino group in the molecule can be mentioned. Since the hydrophilic compound is a low molecule, it does not easily become a steric hindrance to a sugar chain or an antibody, and does not hinder the progress of the sugar chain molecule recognition reaction by the lectin on the target cell membrane surface. Further, the hydrophilic compound does not include a sugar chain to which a lectin used for directing a specific target such as a lectin can be bound in the sugar chain-modified liposome used in the present invention. Examples of such hydrophilic compounds include amino alcohols such as tris (hydroxyalkyl) aminoalkanes including tris (hydroxymethyl) aminomethane, and more specifically, tris (hydroxymethinore) aminoethane. , Tris (hydroxyethyl) aminoethane, tris (hydroxypropyl) aminoethane, tris (hydroxymethyl) aminomethane, tris (hydroxyethyl) aminomethane, tris (hydroxypropyl) aminomethane, tris (hydroxymethyl) aminopropane, tris (hydroxy) And ethyl) aminopropane and tris (hydroxypropyl) aminopropane.

As used herein, “alkyl” refers to a monovalent group formed by losing one hydrogen atom from an aliphatic hydrocarbon (referred to herein as “alkane”) such as methane, ethane, and propane. C _n H _{2n + 1} − (where n is a positive integer). Alkyl can be linear or branched. In the present specification, the “substituted alkyl” refers to an alkyl in which H of the alkyl is substituted with a substituent specified below. Specific examples thereof include C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl. C1-C11 alkyl or C1-C12 alkyl, C1-C2 substituted alkyl, C1-C3 substituted alkyl, C1-C4 substituted alkyl, C1-C5 substituted alkyl, C1-C6 substituted alkyl C1-C7 substituted alkyl, C1-C8 substituted alkyl, C1-C9 substituted alkyl, C1-C10 substituted alkyl, C1-C11 substituted alkyl or C1-C12 substituted alkyl obtain. For alkanes, these specific examples include C1-C2 alkanes, C1-C3 alkanes, C1-C4 alkanes, C1-C5 alkanes, C1-C6 alkanes, C1-C7 alkanes, C1-C8 alkanes, C1-C9 alkanes, C1-C10 alkane, C1-C11 alkane or C1-C12 alkane, C1-C2-substituted alkane, C1-C3-substituted alkane, C1-C4-substituted alkane, C1-C5-substituted alkane, C1-C6 Substituted alkane, C1-C7 substituted alkane, C1-C8 substituted alkane, C1-C9 substituted alkane, C1-C10 substituted alkane, C1-C11 substituted alkane or C1-C12 substituted Alkane. Here, for example, C1-C10 alkyl means linear or branched alkyl having 1 to 10 carbon atoms, methyl (CH _3- ), ethyl (C ₂ H _5- ), n-propyl. _{(CH 3 CH 2 CH 2 -} ), isopropyl _{((CH 3) 2 CH -} ), n- butyl _{(CH 3 CH 2 CH 2 CH} 2 -), n- pentyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 -), n-hexyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 -), n- heptyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 -), n- octyl _(CH 3 CH _{2 CH 2 CH 2 CH 2 CH} 2 CH 2 CH 2 -), n- nonyl _{(CH 3 CH 2 CH 2 CH} 2 CH 2 CH 2 CH 2 CH 2 CH 2 -), n- decyl _(CH 3 CH 2 CH ₂ C _{H 2 CH 2 CH 2 CH 2} CH 2 CH 2 CH 2 -), - C (CH 3) 2 CH 2 CH 2 CH (CH 3) 2, -CH 2 CH (CH 3) such as ₂ are exemplified. In addition, for example, C1-C10 substituted alkyl refers to C1-C10 alkyl, in which one or more hydrogen atoms are substituted with a substituent. As R, C1-C6 alkyl is preferable, and C1-C6 alkyl is particularly preferable.

  When a substance of the invention or a functional group as defined above is substituted by a substituent R, such substituent R is present in one or more and each independently represents hydrogen, alkyl, cycloalkyl, alkenyl, Cycloalkenyl, alkynyl, alkoxy, carbocyclic group, heterocyclic group, halogen, hydroxy, thiol, cyano, nitro, amino, carboxy, acyl, thiocarboxy, amide, substituted amide, substituted carbonyl, substituted Selected from the group consisting of thiocarbonyl, substituted sulfonyl, and substituted sulfinyl.

  Furthermore, a compound obtained by introducing an amino group into a low molecular compound having an OH group can also be used as the hydrophilic compound of the present invention. Although this compound is not limited, For example, the compound which introduce | transduced the amino group into the sugar chain which lectins, such as cellobiose, do not couple | bond is mentioned. For example, the liposome surface is hydrophilized using a divalent reagent for crosslinking and tris (hydroxymethyl) aminomethane on the lipid phosphatidylethanolamine of the liposome membrane. The general formula of the hydrophilic compound is represented by the following formula (1), formula (2), formula (3), and the like.

Z—R ¹ (R ² OH) _n formula (1)
H ₂ N—R ³ — (R ⁴ OH) _n formula (2)
H ₂ N—R ⁵ (OH) _n formula (3)
Here, R ¹ , R ³ and R ⁵ represent a linear or branched hydrocarbon chain of C ₁ to C ₄₀ , preferably C ₁ to C ₂₀ , more preferably C ₁ to C ₁₀ , and R ² , R ⁴ is absent or represents a C ₁ to C ₄₀ , preferably C ₁ to C ₂₀ , more preferably C ₁ to C ₁₀ linear or branched hydrocarbon chain. Z is a reactive functional group bonded to a bivalent reagent for a direct bond or a bridge and liposomal lipid, for _{example, COOH, NH, NH 2,} CHO, SH, NHS- ester, maleimide, imidoesters, active halogens, EDC, Examples include pyridyl disulfide, azidophenyl, hydrazide and the like. n represents a natural number. The surface of the liposome hydrophilized with such a hydrophilic compound is thinly covered with the hydrophilic compound. However, since the thickness of the cover of the hydrophilic compound is small, the reactivity of sugar chains and the like is not suppressed even when sugar chains are bound to liposomes.

  In the present specification, the “hydrophilic compound group” is a name when the above hydrophilic compound is bonded to other groups. The hydrophilic compound group can be monovalent or divalent depending on the case.

  In this specification, the “hydrophilic compound cross-linking group” is a group in which one end is a peptide bond with a linker protein and the other end is a peptide bond with a sugar chain, and between the hydrophilic compound group and the liposome or the linker protein. Refers to a group that forms a peptide bond. Examples of the hydrophilic compound crosslinking group include bis (sulfosuccinimidyl) suberate group, disuccinimidyl glutarate group, dithiobissuccinimidyl propionate group, disuccinimidyl suberate group, 3, Examples thereof include a 3′-dithiobis (sulfosuccinimidylpropionate) group, an ethylene glycol bissuccinimidyl succinate group, and an ethylene glycol bissulfosuccinimidyl succinate group. Preferably, the hydrophilic compound crosslinking group is a bis (sulfosuccinimidyl) suberate group.

  Hydrophilization of liposomes is achieved by a conventionally known method, for example, a method of preparing liposomes using phospholipids obtained by covalently binding polyethylene glycol, polyvinyl alcohol, maleic anhydride copolymer or the like (Japanese Patent Laid-Open No. 2000-302585). (E.g., CNDAC-containing liposomal formulation dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine; dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol; sphingomyelin; cholesterol; -Monomethoxypolyethyleneglycol succinyl-distearoylphosphatidylethanolamine (below PEG2000-DSPE); using CNDAC hydrochloride, glucose aqueous solution and trehalose aqueous solution, the method of Bangham et al. (See J. Mol. Biol. 8, 660-668 (1964).) It can also be carried out by adopting a method such as “A dispersion was obtained”. Among these, it is particularly preferable to make the liposome surface hydrophilic using tris (hydroxymethyl) aminomethane. The method using tris (hydroxymethyl) aminomethane of the present invention is preferable in several respects as compared with the conventional hydrophilization method using polyethylene glycol or the like. For example, in the case where a sugar chain or an antibody or the like is bound on a liposome and its molecular recognition function is used for targeting as in the present invention, tris (hydroxymethyl) aminomethane is a low molecular weight substance, so that conventional polyethylene is used. Compared to a method using a high molecular weight substance such as glycol, it is particularly preferable because it is less likely to cause steric hindrance to the sugar chain and does not hinder the progress of the sugar chain molecule recognition reaction by the lectin (sugar chain recognition protein) on the target cell membrane surface.

  In addition, the liposome used in the present invention has a good particle size distribution, component composition, and dispersion characteristics even after the hydrophilization treatment, and has excellent long-term storage and in vivo stability. It is preferable for use. In order to hydrophilize the liposome surface using tris (hydroxymethyl) aminomethane, for example, lipids such as dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine were obtained by a conventional method. Bissulfosuccinimidyl suberate, disuccinimidyl glutarate, dithiobissuccinimidyl propionate, disuccinimidyl suberate, 3,3′-dithiobis (sulfosuccinimidyl propionate) , Dipalmitoylphosphatidyl on the liposome membrane by adding and reacting a divalent reagent such as ethylene glycol bissuccinimidyl succinate, ethylene glycol bissulfosuccinimidyl succinate By binding a divalent reagent to a lipid such as ethanolamine, and then reacting tris (hydroxymethyl) aminomethane with the negative bond of the divalent reagent, tris (hydroxymethyl) aminomethane is bound to the liposome surface. Let them join together.

  As described above, the hydrophilized liposome is extremely stable in vivo, and has a long half-life in vivo without binding to a target-directed sugar chain or antibody as described later. It can be suitably used as a drug carrier in a drug delivery system. The liposome used in the present invention also includes a liposome whose surface is rendered hydrophilic with a low molecular weight compound.

  As used herein, “delivery vehicle” refers to a carrier (vehicle) that mediates delivery of a desired substance. If the substance to be delivered is a drug, it is referred to as a “drug delivery vehicle”. The drug delivery system (Drug Delivery System, DDS) is also called a drug delivery system, and is sometimes classified into an absorption-controlled DDS, a controlled-release DDS, and a target-oriented DDS. An ideal DDS is a system that delivers a drug “to the necessary site in the body”, “a necessary amount”, and “for the required time”. Targeting DDS (written as Targeting DDS, translated into target-oriented DDS) is categorized as passive targeting (active) and active targeting (active). . The former is a method of controlling the behavior in the body using physicochemical properties such as the particle size and hydrophilicity of the carrier (drug carrier). The latter is a method in which a special mechanism is added to these to actively control the directivity to the target tissue. For example, an antibody having a specific molecule recognition function for a target molecule of a specific cell constituting the target tissue There is a method using a carrier to which a sugar chain or the like is bound, and it is sometimes called a “missile drug”.

  As used herein, “drug delivery vehicle” refers to a vehicle for delivering a desired drug.

  In another aspect, the pharmaceutical composition of the present invention can also be used as a molecular imaging agent. The molecular imaging agent can further include a pharmaceutically acceptable carrier and the like. Pharmaceutically acceptable carriers include, for example, antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, extenders, buffers, delivery vehicles, diluents Examples include, but are not limited to, agents, excipients and / or pharmaceutical adjuvants. Typically, the molecular imaging agent is administered in the form of a composition comprising a pharmaceutical composition of the invention, one or more physiologically acceptable carriers, excipients or diluents. For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid.

  Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used. Such non-toxic and inert carriers include, for example, phosphate, citrate, or other organic acids; ascorbic acid, α-tocopherol; low molecular weight polypeptides; proteins (eg, serum albumin, gelatin or Immunoglobulins); hydrophilic polymers (eg polyvinylpyrrolidone); amino acids (eg glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrin); (Eg, EDTA); sugar alcohols (eg, mannitol or sorbitol); salt-forming counterions (eg, sodium); and / or non-ionic surface active agents (eg, Tween, pluroni). ) Or polyethylene glycol (PEG)) but the like are not limited thereto.

  Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include pH 7.0-8.5 tris (hydroxymethyl) aminomethane buffer or pH 4.0-5.5 acetate buffer, which further includes sorbitol or suitable thereof Alternatives may be included.

  In one aspect, the present invention provides a pharmaceutical composition that can be used as a molecular imaging agent including a sugar chain-modified liposome. The drug delivery vehicle of the present invention binds a sugar chain having at least one structure selected from the group consisting of Gal, GalNAc, Man, Glc, GlcNAc, Fuc and Neu5Ac, preferably a sugar chain shown in Table 1 above Sugar chain-modified liposomes. The pharmaceutical composition may further encapsulate or bind a drug or gene.

  As used herein, “molecular imaging agent” refers to a drug or factor used to image a function or structure of a living body. Examples thereof include those used for imaging arteriosclerotic lesions or unstable plaques in vivo.

  The molecular imaging agent of the present invention is used for administering a biological agent to a subject in need of the biological agent, and for the respiratory system, circulatory system, digestive system, urogenital / genital system, central nervous system or It can also be used to treat mammals with peripheral nervous system disorders.

  The molecular imaging agent of the present invention can also enhance the absorption controllability in the intestinal tract by adjusting the type of sugar chain bound to the liposome and the binding density. By binding both a sugar chain that enhances intestinal absorption controllability and a sugar chain or antibody that has directivity to specific tissues or organs to liposomes, both directivity to specific tissues or organs and intestinal absorption controllability are combined. It is also possible to produce liposomes having the above characteristics.

  In the sugar chain-modified liposome that can be contained in the molecular imaging agent of the present invention, the sugar chain can be a sialyl Lewis X group. This sialyl Lewis X group can be contained in a sugar chain-modified liposome at a modified bond density of 0.0001 mg sugar chain / mg lipid to 500 mg sugar chain / mg lipid. When imaging an arteriosclerotic lesion, preferably, a sugar chain-modified liposome with a modified bond density of 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid) Can be included. When imaging unstable plaques, it is preferred that the liposome be modified with a modified binding density of 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid). Can be included.

  In the sugar chain-modified liposome that can be contained in the molecular imaging agent of the present invention, the sugar chain can be an α1-6 mannobiose group. The α1-6 mannobiose group can be contained in the sugar chain-modified liposome at a modified bond density of 0.0001 mg sugar chain / mg lipid to 500 mg sugar chain / mg lipid. When imaging macrophages, macrophages-related diseases, disorders or conditions, preferably 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid). It can be contained in a sugar chain-modified liposome at a modified bond density.

  The molecular imaging agent of the present invention can be used to image atherosclerotic lesions, macrophages, macrophage related diseases, disorders or. These tissues may contain parenchyma.

  The molecular imaging agent of the present invention can be easily prepared by those skilled in the art by considering pH, isotonicity, stability, and the like. The molecular imaging agent of the present invention is blended with a pharmaceutically acceptable carrier and orally as a liquid preparation such as a solid preparation such as a tablet, capsule, granule, powder or powder, a syrup, a suspension or a solution. Can be administered.

  The molecular imaging agent of the present invention may contain a physiologically acceptable carrier, excipient, or stabilizer as necessary (Japanese Pharmacopoeia 14th edition or its latest edition, Remington's Pharmaceutical Sciences, 18th Edition, A.M. R. Gennaro, ed., Mack Publishing Company, 1990, etc.) and a glycan composition having the desired degree of purity are mixed and prepared in the form of a lyophilized cake or aqueous solution. obtain.

  By incorporating a liposome containing a fluorescent dye and modified with a sugar chain or an antibody into the pharmaceutical composition of the present invention, it is possible to perform imaging with higher sensitivity than conventional imaging agents. This is because, by selecting a fluorescent dye having an excitation and fluorescence detection wavelength of 500 to 700 nm in a long wavelength, it can be distinguished from autofluorescence derived from biological components, so that highly sensitive imaging from outside the living body can be realized. This is because it becomes possible.

  The amount of the molecular imaging agent used in the treatment method of the present invention takes into consideration the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, and the like. Thus, it can be easily determined by those skilled in the art. The frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  Molecular imaging agents can be formulated using pharmaceutically acceptable carriers well known in the art in dosage forms suitable for administration. Such carriers allow drug delivery vehicles to be formulated into liquids, gels, syrups, slurries, suspensions, etc. suitable for ingestion by patients.

  In one aspect, the present invention provides a carrier for use in molecular or in vivo imaging. This carrier comprises a pharmaceutical composition comprising hydrophilized liposomes and a specific factor for arteriosclerotic lesions. The hydrophilic liposome may be modified with a sugar chain or an antibody, and may further contain a labeling substance.

  In the sugar chain-modified liposome that can be contained in the carrier of the present invention, the sugar chain can be a sialyl Lewis X group. The sialyl Lewis X group can be contained in the sugar chain-modified liposome with a modified bond density of 0.0001 mg sugar chain / mg lipid to 500 mg sugar chain / mg lipid. When imaging an arteriosclerotic lesion, preferably, a sugar chain-modified liposome with a modified bond density of 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid) Can be included. When imaging unstable plaques, it is preferred that the liposome be modified with a modified binding density of 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid). Can be included.

  In the sugar chain-modified liposome that can be contained in the carrier of the present invention, the sugar chain can be an α1-6 mannobiose group. The α1-6 mannobiose group can be included in the sugar chain-modified liposome at a modified bond density of 0.0001 mg sugar chain / mg lipid to 500 mg sugar chain / mg lipid. When imaging macrophages, macrophages-related diseases, disorders or conditions, preferably 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid). It can be contained in a sugar chain-modified liposome at a modified bond density.

  The vehicle or pharmaceutical composition of the present invention includes a composition in which the fluorescent dye, drug or biological agent is contained in the hydrophilized liposomes in an amount effective to achieve the intended purpose. “An amount effective to treat” is a term well recognized by those skilled in the art and refers to an amount of an agent effective to produce an intended pharmacological result (eg, prevention, treatment, prevention of recurrence, etc.). Say. Thus, a therapeutically effective amount is an amount sufficient to reduce the symptoms of the disease to be treated. One useful assay to ascertain an effective amount (eg, a therapeutically effective amount) for a given application is to measure the extent of recovery of the target disease. The amount actually administered will depend on the individual to whom the treatment is to be applied, and is preferably an amount optimized to achieve the desired effect without significant side effects. The determination of an effective dose is well within the ability of those skilled in the art.

Therapeutically effective amount, prophylactically effective amount and the like and toxicity are standard pharmaceutical procedures in cell cultures or experimental animals (eg ED ₅₀ , therapeutically effective dose in 50% of the population; and LD ₅₀ , 50% of the population. The dose that is lethal to). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as ratio ED 50 _/ LD _50. Drug delivery vehicles that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal experiments are used to formulate a range of quantities for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. This dose will vary within this range depending on the mode of administration used, the sensitivity of the patient, and the route of administration. As an example, the dose is appropriately selected depending on the age and other patient conditions, the type of disease, the type of cells used, and the like.

  The compositions, molecular imaging agents, media, etc. of the present invention can be manufactured in a manner similar to that known in the art (eg, mixing, dissolution, etc.).

  The composition of the present invention for delivering a substance to a desired site may include a liposome modified with a sugar chain or an antibody (antibody, antibody fragment, antibody variant). In the sugar chain-modified liposome that can be contained in the composition of the present invention, the sugar chain can be a sialyl Lewis X group. This sialyl Lewis X group can be contained in a sugar chain-modified liposome at a modified bond density of 0.0001 mg sugar chain / mg lipid to 500 mg sugar chain / mg lipid. When imaging an arteriosclerotic lesion, preferably, a sugar chain-modified liposome with a modified bond density of 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid) Can be included. When imaging unstable plaques, it is preferred that the liposome be modified with a modified binding density of 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid). Can be included.

  In the sugar chain-modified liposome that can be contained in the composition of the present invention, the sugar chain may be an α1-6 mannobiose group. The α1-6 mannobiose group can be contained in the sugar chain-modified liposome at a modified bond density of 0.0001 mg sugar chain / mg lipid to 500 mg sugar chain / mg lipid. When imaging macrophages, macrophages-related diseases, disorders or conditions, preferably 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid). It can be contained in a sugar chain-modified liposome at a modified bond density.

  In the present specification, the “instruction” refers to a method for administering the pharmaceutical composition, diagnostic composition or molecular imaging agent of the present invention, etc. ). This instruction describes a word indicating a procedure for administering the pharmaceutical composition, diagnostic composition, molecular imaging agent, or the like of the present invention. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction sheet is a so-called package insert, and is usually provided as a paper medium, but is not limited thereto. For example, an electronic medium (for example, a home page (web site) or e-mail provided on the Internet) It can also be provided in such a form.

(Composition)
(Pharmaceutical composition)
In one aspect, the present invention provides a pharmaceutical composition for diagnosing, treating or preventing arteriosclerotic lesions, the pharmaceutical composition comprising hydrophilicized liposomes and a specific factor for arteriosclerotic lesions. In this case, any form described in the above (Liposome) can be used as the hydrophilicized liposome.

  In one embodiment, in the pharmaceutical composition of the present invention, the specific factor for the atherosclerotic lesion is sialyl Lewis X (SLX), SLX group, mannose, mannose group, antibody (eg, VCAM-1, ICAM-1, And antibodies to cell adhesion molecules such as E-selectin and P-selectin, antibodies to extracellular matrix-degrading enzymes such as MMP), antibody fragments, and antibody variants, but are not limited thereto. As long as it is a specific factor for arteriosclerotic lesions, it is possible to increase the accumulation of the pharmaceutical composition in the arteriosclerotic lesions. Can do. Preferably, the specific factor for the atherosclerotic lesion can be sialyl Lewis X (SLX) or SLX group.

  In another embodiment, in the pharmaceutical composition of the invention, the diagnosis is a diagnosis of vulnerable plaque.

  In another embodiment, in the pharmaceutical composition of the present invention, the hydrophilization can be performed by amino alcohols such as tris (hydroxyalkyl) aminoalkane. More specifically, tris (hydroxymethinore) aminoethane, tris (hydroxyethyl) aminoethane, tris (hydroxypropyl) aminoethane, tris (hydroxymethyl) aminomethane, tris (hydroxyethyl) aminomethane, tris (hydroxypropyl) amino Examples include methane, tris (hydroxymethyl) aminopropane, tris (hydroxyethyl) aminopropane, tris (hydroxypropyl) aminopropane, and the like. Preferably, the hydrophilization can be done with a tris (hydroxymethyl) methylamino group.

  In another embodiment, in the pharmaceutical composition of the present invention, a part of the lipid constituting the liposome is a hydrophilic compound cross-linking group -W-tris (hydroxymethyl) methylamino group and NH-C (= O) bond. Where W is a C (═O) —NH bond. Examples of lipids constituting the liposome include phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, long-chain alkyl phosphates, glycolipids (gangliosides, etc.), phosphatidylglycerols, sphingomyelins, cholesterols and the like. Can be mentioned.

  In another embodiment, in the pharmaceutical composition of the present invention, the liposome and the SLX group may be bound via a linker group. As the linker group, a group derived from a biological protein, particularly a human derived protein is preferably used. The group of a protein derived from a living body is not limited, and examples thereof include a group such as a protein present in blood such as albumin and other physiologically active substances present in a living body. For example, there are animal serum albumin groups such as human serum albumin (HSA) group and bovine serum albumin (BSA) group. Especially when human serum albumin group is used, there is a lot of uptake into each tissue. It has been confirmed by the experiment.

In a preferred embodiment, in the pharmaceutical composition of the present invention, a part of the lipid constituting the liposome has a human serum albumin group-A-linker protein cross-linking group-X-SLX group and a CH ₂ -NH bond, Here, the A may be an NH—C (═O) bond, and the X may be a C (═O) —NH bond.

  In another preferred embodiment, in the pharmaceutical composition of the present invention, the SLX group and the antibody group are present on the surface of the liposome.

  In another aspect, the present invention provides a pharmaceutical composition for diagnosing, treating or preventing a macrophage or a disease, disorder or condition associated with macrophage. The pharmaceutical composition comprises hydrophilicized liposomes and specific factors for macrophages such as mannose, mannose groups, antibodies, antibody fragments and antibody variants.

  In one embodiment, in the pharmaceutical composition of the present invention, the diagnosis, treatment or prevention may include targeting only atherosclerotic lesions in which highly active macrophages are present. Here, any form described in the above-mentioned (Liposome) can be used as the hydrophilicized liposome.

(Diagnostic composition)
In another aspect, the present invention provides a diagnostic composition for diagnosing arteriosclerotic lesions. This diagnostic composition comprises A) a hydrophilic liposome, B) a specific factor for arteriosclerotic lesions, and C) a labeling substance, wherein the hydrophilic liposome is the above-mentioned (liposome). ) And any form described in (Pharmaceutical compositions) may be used.

  In one embodiment, in the diagnostic composition of the present invention, the specific factor for atherosclerotic lesion is sialyl Lewis X (SLX), SLX group, mannose, mannose group, antibody (eg, VCAM-1, ICAM-1). , Antibodies against cell adhesion molecules such as E-selectin and P-selectin, antibodies against extracellular matrix degrading enzymes such as MMP, etc.), antibody fragments, antibody variants and the like. As long as it is a factor specific to vulnerable plaque, it is possible to increase the accumulation of the diagnostic composition in the arteriosclerotic lesion, so it is preferable to use anything as long as there is no toxicity Can do. Preferably, the specific factor for the atherosclerotic lesion can be sialyl Lewis X (SLX) or SLX group.

  In other embodiments, the diagnostic compositions of the present invention can be used in magnetic resonance imaging.

  In another embodiment, in the diagnostic composition of the present invention, the diagnosis can be made by diagnosis of vulnerable plaque.

  In another embodiment, the diagnostic composition of the present invention can be used to diagnose arteriosclerotic lesions by magnetic resonance imaging, wherein the labeling substance is detectable by nuclear magnetic resonance imaging. It is a substance. This labeling substance is a paramagnetic metal (eg, gadolinium, iron, etc.), a contrast agent (eg, iron oxide particles, gadolinium chelating agent, barium sulfate, water-soluble iodine, iohexol, iopamidol, urografin, etc.), ultrasonic contrast It can be applied by an agent (for example, levovist, sonazoid, etc.), but is not limited thereto. Preferably, the labeling substance is a paramagnetic metal.

  In another aspect, the present invention provides a diagnostic composition for diagnosing macrophages or macrophage-related diseases, disorders or conditions. This diagnostic composition comprises A) a hydrophilic liposome, B) a mannose or mannose group, and C) a labeling substance, wherein the hydrophilic liposome is described in (Liposome) above. Any form can be used.

  In one embodiment, in the diagnostic composition of the present invention, the diagnosis may include diagnosing only an arteriosclerotic lesion in which highly active macrophages are present.

  The compositions of the present invention can optionally include a suitable formulation material or a pharmaceutically acceptable carrier. Suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, fluorescent dyes, flavoring agents, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffering agents. , Delivery vehicles and / or pharmaceutical adjuvants. Typically, the composition of the present invention comprises sialyl Lewis X (SLX), and optionally other active ingredients, together with at least one physiologically acceptable carrier, excipient or diluent. It is administered in the form of For example, suitable vehicles can be micelles, injection solutions, physiological solutions, or artificial cerebrospinal fluid, which are supplemented with other substances commonly used in compositions for parenteral delivery. It is possible.

  Acceptable carriers, excipients or stabilizers used herein are preferably non-toxic to recipients and preferably inert at the dosages and concentrations used. Preferably, for example, phosphate, citrate, or other organic acid; ascorbic acid, α-tocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer (eg, Amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, EDTA); Mannitol or sorbitol ); Salt-forming counterions (such as sodium); and / or non-ionic surface-active agents (e.g., Tween, Pluronic (pluronic) or polyethylene glycol (PEG)), and the like.

  Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include a Tris buffer with a pH of about 7.0-8.5 or an acetate buffer with a pH of about 4.0-5.5, which further includes sorbitol or a suitable replacement thereof. Can contain things.

  The general preparation method of the composition of this invention is shown below. It should be noted that veterinary drug compositions, quasi-drugs, marine drug compositions, food compositions, cosmetic compositions and the like can also be produced by known preparation methods.

  The composition of the present invention may be blended with a pharmaceutically acceptable carrier as necessary, and administered parenterally, for example, as a liquid preparation such as an injection, suspension, solution, spray or the like. it can. Examples of pharmaceutically acceptable carriers include excipients, lubricants, binders, disintegrants, disintegration inhibitors, absorption enhancers, absorbents, wetting agents, solvents, solubilizers, suspending agents, Examples include isotonic agents, buffering agents, soothing agents and the like. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used as necessary. Moreover, it is also possible to mix | blend substances other than sialyl Lewis X, a lipid, etc. with the composition of this invention. Examples of parenteral administration routes include, but are not limited to, intravenous, intramuscular, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, topical administration, and dermal administration. When administered systemically, the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens. It is within the skill of the artisan to consider pH, isotonicity, stability, etc. for the preparation of such pharmaceutically acceptable compositions.

  Preferable examples of the solvent in the liquid preparation include injection solutions, alcohol, propylene glycol, macrogol, sesame oil and corn oil.

  Preferable examples of the solubilizer in the liquid preparation include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. It is not limited to them.

  Suitable examples of the suspending agent in the liquid preparation include, for example, surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, Examples thereof include hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose.

  Preferable examples of the isotonic agent in the liquid preparation include, but are not limited to, sodium chloride, glycerin, D-mannitol and the like.

  Preferable examples of the buffer in the liquid preparation include, but are not limited to, phosphate, acetate, carbonate, citrate and the like.

  Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalkonium chloride, procaine hydrochloride and the like.

  Preferable examples of the preservative in the liquid preparation include, but are not limited to, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, sorbic acid and the like.

  Preferable examples of the antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, α-tocopherol and cysteine.

  When prepared as injections, solutions and suspensions are preferably sterilized and isotonic with solvents in the injection site for blood or other purposes. Usually, these are sterilized by filtration using a bacteria retention filter or the like, blending with a bactericide, or irradiation. Furthermore, after these treatments, solidify by freeze-drying or other methods, and add sterile water or sterile diluent for injection (lidocaine hydrochloride aqueous solution, physiological saline, aqueous glucose solution, ethanol, or a mixed solution thereof) just before use. May be.

  Furthermore, the composition of this invention may contain the coloring agent, the preservative, the fragrance | flavor, the flavoring agent, the sweetener, etc., and another chemical | medical agent.

  The amount of the substance, composition, etc. used in the present invention takes into consideration the purpose of use, the target disease (type, severity, etc.), the age, weight, sex, past history, cell morphology or type of the subject. Thus, it can be easily determined by those skilled in the art. The frequency with which the method of the present invention is applied to a subject also takes into account the purpose of use, the target disease (type, severity, etc.), the subject's age, weight, gender, medical history, treatment course, etc. A person skilled in the art can easily determine. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course. The amount to be administered can be determined by estimating the amount required by the site to be treated.

  In this specification, “Magnetic Resonance Imaging (MRI)” refers to a method of imaging internal information in a living body using a nuclear magnetic resonance phenomenon.

  In this specification, “nuclear magnetic resonance (NMR)” means that resonance occurs between an oscillating magnetic field or electromagnetic waves when the spin energy levels of nuclei are separated by the Zeeman effect.

  In the present specification, the “contrast agent” refers to a drug used for increasing or decreasing the concentration (signal intensity) of a tissue in diagnostic imaging. What is increased is called a positive contrast agent, and what is decreased is called a negative contrast agent. Examples of positive contrast agents include, but are not limited to, gadolinium chelating agents, barium sulfate, and water-soluble iodine contrast agents. Negative contrast agents include, but are not limited to, superparamagnetic iron oxide particles (SPIO), air, carbon dioxide, and the like. Examples of the ultrasound contrast agent may include levovist and sonazoid.

  As used herein, “subject” refers to an animal that is the subject of treatment, detection or diagnosis of ischemic brain disease, ischemic brain injury or cerebral ischemic condition. The animals targeted by the present invention may be, for example, birds and mammals. Preferably, such animals are mammals (eg, single pores, marsupials, rodents, wings, wings, carnivorous, carnivorous, long-nosed, odd-hoofed, cloven-hoofed) , Rodents, scales, sea cattle, cetaceans, primates, rodents, rabbits, etc.). Exemplary subjects include, but are not limited to, animals such as cows, pigs, horses, chickens, cats, dogs and the like. More preferably, small animals such as mice, rats, rabbits, hamsters and guinea pigs can be used.

(Use of specific factor-containing compounds for arteriosclerotic lesions as pharmaceuticals)
In another aspect, the present invention provides the use of a compound containing a specific factor for arteriosclerotic lesions in the manufacture of a medicament for treating, preventing, detecting or diagnosing arteriosclerotic lesions.

  In another aspect, the compound containing a specific factor for arteriosclerotic lesion of the present invention can be used as a medicament. In such a case, examples of the drug encapsulated or bound to the specific factor-containing compound include, but are not limited to, the following: alkylated anticancer agents, antimetabolites, plant-derived anticancer agents, anticancer properties Antibiotics, BRM / cytokines, platinum complex anticancer agents, immunotherapy agents, hormonal anticancer agents, tumor drugs such as monoclonal antibodies, drugs for central nervous system, drugs for peripheral nervous system / sensory organs, drugs for treating respiratory diseases, circulation Drugs for genital organs, drugs for digestive organs, drugs for hormones, drugs for urology / genital organs, vitamins / nutritional tonics, metabolic drugs, antibiotics / chemotherapeutic drugs, drugs for testing, anti-inflammatory drugs, drugs for eye diseases, central nervous system Drugs, autoimmune drugs, cardiovascular drugs, lifestyle-related diseases such as diabetes and hyperlipidemia, corticosteroids, immunosuppressants, antibacterial drugs, antiviral drugs, angiogenesis Inhibitors, cytokines, chemokines, anti-cytokine antibodies, anti-chemokine antibodies, anti-cytokine / chemokine receptor antibodies, siRNAs, miRNAs, smRNAs, gene therapy-related nucleic acid preparations such as antisense ODNs or DNAs, neuroprotective factors, antibody drugs Drugs such as molecular targeted drugs, osteoporosis / bone metabolism improving drugs, neuropeptides, bioactive peptides / proteins.

(Imaging method)
In another aspect, the present invention provides a method for imaging arteriosclerotic lesions. The method includes administering to a subject a molecular imaging agent, the molecular imaging agent comprising a pharmaceutical composition of the present invention, wherein the pharmaceutical composition is A) hydrophilized. The composition comprises a liposome, B) a specific factor for arteriosclerotic lesion, and C) a labeling substance, and the pharmaceutical composition contains a sufficient amount of the labeling substance for detection. Here, as the pharmaceutical composition, any form described in (Liposome), (Composition), and (Pharmaceutical composition) described above can be used.

(Imaging system)
In another aspect, the present invention provides a system for molecular or in vivo imaging of arteriosclerotic lesions. As used herein, “molecular imaging” or “in vivo imaging” refers to imaging a function or structure of a living body. The system for molecular or in vivo imaging of the present invention comprises the pharmaceutical composition of the present invention and a means for examining the presence or absence of the label, wherein the pharmaceutical composition comprises: A) a hydrophilized liposome; B) a specific factor for arteriosclerotic lesions and C) a labeling substance, wherein after a sufficient time for the label to accumulate in the arteriosclerotic lesion, the presence or absence of the label in the living body is examined, The function or structure of the living body can be imaged by the label. Examples of the label include, but are not limited to, a fluorescent substance, a radioactive substance, a coloring substance (for example, βgal), an issuing substance (for example, luciferase), a paramagnetic metal, and the like.

  The sugar chain of the sugar chain-modified liposome that can be used in the system of the present invention can be a sialyl Lewis X group. This sialyl Lewis X group can be contained in a sugar chain-modified liposome at a modified bond density of 0.0001 mg sugar chain / mg lipid to 500 mg sugar chain / mg lipid. When imaging an arteriosclerotic lesion, preferably, a sugar chain-modified liposome with a modified bond density of 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid) Can be included. When imaging unstable plaques, it is preferred that the liposome be modified with a modified binding density of 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid). Can be included.

  The sugar chain of the sugar chain-modified liposome that can be used in the system of the present invention may be an α1-6 mannobiose group. The α1-6 mannobiose group can be contained in the sugar chain-modified liposome at a modified bond density of 0.0001 mg sugar chain / mg lipid to 500 mg sugar chain / mg lipid. When imaging macrophages, macrophages-related diseases, disorders or conditions, preferably 0.0025 mg sugar chain / mg lipid to 0.1 mg sugar chain / mg lipid (preferably 0.025 mg sugar chain / mg lipid). It can be contained in a sugar chain-modified liposome at a modified bond density.

  With the system of the present invention, it is possible to image atherosclerotic lesions, unstable plaques, or sites of diseases, disorders or conditions related to macrophages. These tissues may contain parenchyma. The means for examining the presence or absence of a label used in the system of the present invention may be a scanning microscope. Preferably, the means for checking the presence or absence of the label further includes a stick objective lens. As long as the label can be detected, any substance (for example, fluorescence, radiation, color developing substance (for example, βgal), issuing substance (for example, luciferase), paramagnetic metal, etc.) can be detected. Or other means). This is because the function or structure of the living body can be imaged by detecting the label.

(Treatment method)
In another aspect, the present invention provides a method for treating or preventing arteriosclerotic lesions. This method involves administering an effective amount of a pharmaceutical composition of the present invention to a patient in need of treating or preventing an arteriosclerotic lesion. Here, the pharmaceutical composition contains hydrophilic liposomes and a specific factor for arteriosclerotic lesions. As the hydrophilized liposome, any form described in the above (Liposome) can be used.

(Diagnosis method)
In another aspect, the present invention provides a method for detecting arteriosclerotic lesions. This method comprises the steps of administering an effective amount of a diagnostic composition to a patient in need of detecting an arteriosclerotic lesion; and measuring the labeling substance contained in the diagnostic composition in the patient by magnetic resonance imaging. And the presence of the labeling substance includes a step indicating that the patient has an arteriosclerotic lesion, and the diagnostic composition comprises A) a hydrophilized liposome, and B) specific to an arteriosclerotic lesion. A factor and C) a labeling substance. Here, any form described in the above-mentioned (Liposome) can be used as the hydrophilicized liposome.

(Method for producing sugar chain or antibody-modified liposome containing labeling substance)
The method for producing a sugar chain or antibody-modified liposome containing a labeling substance used in the present invention includes, for example, A) a step of forming a liposome in which a labeling substance is encapsulated or bound; B) the liposome And C) a step of binding the liposome to a linker protein, and D) a method of binding a sugar chain or an antibody to the liposome.

  In another embodiment, a method for producing a sugar chain or antibody-modified liposome containing a labeling substance used in the present invention comprises: (a) suspending a lipid in a methanol / chloroform solution and stirring the solution. Evaporating the precipitate and vacuum drying the precipitate to obtain a lipid membrane; (b) suspending the lipid membrane in a suspension buffer and sonicating; (c) the sonicated solution and Mixing with a fluorescent labeling solution to provide fluorescently labeled liposomes; (d) hydrophilizing the liposomes with tris (hydroxyalkyl) aminoalkane; (e) hydrophilizing A step of binding a linker protein to the liposome to form a linker protein-bound liposome; and (f) a sugar chain by binding a sugar chain or an antibody to the liposome. Other includes the step of generating an antibody-modified liposome. Here, any form described in the above (Liposome) can be used as the liposome.

  In the present invention, a liposome modified with a sugar chain or an antibody used in a pharmaceutical composition or a diagnostic composition for delivering a substance to a target delivery site can be produced, for example, by the following method: (a) Providing fluorescently labeled sugar chains or antibody-modified liposomes having various sugar chains or antibody densities to achieve delivery to the intended delivery site, comprising: (i) lipids in methanol / chloroform Suspending in a solution and stirring, evaporating the stirred solution and drying the precipitate in vacuo to obtain a lipid membrane; (ii) suspending the lipid membrane in a suspension buffer and sonicating (Iii) mixing the sonicated solution and the fluorescent labeling solution; (b) for the sugar chain density on the sugar chain or antibody-modified liposome to the delivery site; Determining the density to achieve proper delivery; and (c) incorporating the substance (eg, fluorescent dye, drug) into the determined optimal sugar chain or antibody-modified liposome to produce a drug-containing liposome. To do. Incorporation of a substance (for example, fluorescent dye, medicine) can include, for example, encapsulation, binding to an outer surface, and the like. Here, as the sugar chain or sugar chain-modified liposome, any form described in the above (Liposome) can be used.

(Binding or encapsulation of labeling substance)
As for the binding or encapsulation of the labeling substance, any of those used for binding or encapsulating drugs in liposomes is used. For example, (1) dipalmitoyl phosphatidylcholine (DPPC), cholesterol, ganglioside, dicetyl phosphate (DCP), dipalmitoyl phosphatidylethanolamine (DPPE), sodium cholate are weighed, and methanol / chloroform solution ( 1: 1) and stirred for 1 hour at 37 ° C .; (2) chloroform / methanol is evaporated on a rotary evaporator and dried in vacuum. Resuspended in N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), stirred at 37 ° C. for 1 hour, purged with nitrogen, and sonicated. (3) Sonication The solution and fluorescent dye (for example, cy5.5, cy3, cy7) labeled human serum albumin solution (fluorescent dye-labeled HSA solution) are mixed and subjected to ultrafiltration (fractionated molecular weight: 10,000) to label HSA-encapsulated liposome particles labeled with a substance (for example, a fluorescent dye (for example, cy5.5, cy3, cy7), paramagnetic metal, etc.) are obtained.

(Manufacture of liposomes)
The liposome itself can be produced according to a known method, and examples thereof include a thin film method, a reverse layer evaporation method, an ethanol injection method, and a dehydration-rehydration method.

  Moreover, it is also possible to adjust the particle diameter of the liposome using an ultrasonic irradiation method, an extrusion method, a French press method, a homogenization method, or the like. The production method of the liposome itself used in the present invention will be specifically described. For example, first, phosphatidylcholines, cholesterol, phosphatidylethanolamines, phosphatidic acids, gangliosides, glycolipids, or phosphatidylglycerols are used as components. Prepare mixed micelles of lipids and surfactant sodium cholate. In particular, the formulation of long-chain alkyl phosphates such as phosphatidic acids or dicetyl phosphate is essential for negatively charging the liposomes, and the formulation of phosphatidylethanolamines is used as a hydrophilization reaction site as gangliosides or sugars. Formulation of lipids or phosphatidylglycerols is essential as a linker binding site. At least one lipid selected from the group consisting of gangliosides, glycolipids, phosphatidylglycerols, sphingomyelins and cholesterols assembles in the liposome and functions as a scaffold (raft) to which the linker is bound. Liposomes used in the present invention are further stabilized by the formation of rafts that can bind such proteins. That is, the liposome used in the present invention is formed with a raft of at least one lipid selected from the group consisting of gangliosides, glycolipids, phosphatidylglycerols, sphingomyelins and cholesterols for binding a linker. Liposomes. And a liposome is produced by performing ultrafiltration of the mixed micelle obtained by this. The liposome used in the present invention may be a normal one, but it is desirable that its surface be made hydrophilic. After the liposome is produced as described above, the liposome surface is made hydrophilic.

  The present invention also includes liposomes that are not bound to sugar chains that have been rendered hydrophilic using the above-mentioned hydrophilic compound. Such hydrophilic liposomes have the advantage that the stability of the liposome itself is enhanced, and that the sugar chain is recognizable when the sugar chain is bound. Liposomes used in the present invention include, for example, liposomes having phosphatidylcholines (molar ratio 0 to 70%), phosphatidylethanolamines (molar ratio 0 to 30%), phosphatidic acids, and long-chain alkyl phosphates. And one or more lipids selected from the group consisting of and dicetyl phosphates (molar ratio 0-30%), one or more selected from the group consisting of gangliosides, glycolipids, phosphatidylglycerols and sphingomyelins Liposome (containing a molar ratio of 0 to 40%) and cholesterol (molar ratio of 0 to 70%).

  The present invention further includes a method of making the liposome hydrophilic by binding the hydrophilic compound to the liposome. Further, hydrophilic liposomes to which sugar chains are not bonded are also included. The target-directed liposome or intestinal absorbable liposome of the present invention can be produced by binding a sugar chain to a liposome to which no sugar chain is bound.

Preferably, the hydrophilization can be performed as follows. (1) Perform ultrafiltration (fractionated molecular weight: 300,000) in order to exchange the buffer with a carbonate buffer (50 mM NaHCO ₃ , 157 mM NaCl, pH 8.5). (2) Add the cross-linking agent BS ³ (PIERCE), and after 2 hours at room temperature, stir overnight under refrigeration. (3) Perform ultrafiltration (fraction molecular weight: 300,000) to remove free BS ³ . (4) Add tris (hydroxymethyl) aminomethane / carbonate buffer (pH 8.5) solution, and after 2 hours at room temperature, stir overnight under refrigeration. (5) Free tris (hydroxymethyl) aminomethane was removed, and ultrafiltration (fractionated molecular weight) was performed for buffer exchange with N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4). : 300,000).

More preferably, the hydrophilization can be performed as follows. (1) In order to exchange the buffer with a carbonate buffer (CBS buffer: 50 mM NaHCO _3, 157 mM NaCl (pH 8.5)), ultrafiltration (fractionated molecular weight: 100,000 _, 2000 × g for 60 minutes) 2 Implement once. (2) Add the cross-linking agent BS ³ (PIERCE), and after 2 hours at room temperature, stir overnight under refrigeration. (3) In order to remove free BS ³ , ultrafiltration (fractionated molecular weight: 100,000, 60 minutes at 2000 × g) is performed twice. (4) Add tris (hydroxymethyl) aminomethane / CBS buffer (pH 8.5) solution, and after 2 hours at room temperature, stir overnight under refrigeration. (5) Free tris (hydroxymethyl) aminomethane was removed, and ultrafiltration (fractionated molecular weight) was performed for buffer exchange with N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4). : 100,000, 2000 × g for 60 minutes) twice.

  In one embodiment, the method for producing a liposome used in the present invention comprises: a) providing a liposome encapsulating fluorescence to which a linker protein is bound; b) hydrophilizing the liposome; c) 3 , 3′-dithiobis (sulfosuccinimidyl propionate) is bound to the liposome; and d) a sugar chain or an antibody is bound to the linker protein in the liposome, and the sugar chain contains the fluorescent dye. Alternatively, the method includes a step of generating an antibody-modified liposome, and the steps b) to c) can be performed in any order. In this embodiment, e) the step of hydrophilizing the sugar chain or antibody-modified liposome containing the fluorescent dye; f) the solution containing the sugar chain or antibody-modified liposome containing the hydrophilic fluorescent dye being filtered A step of filtering can further be included. As the sugar chain or antibody-modified liposome, any form described in the above (Liposome) can be used.

  In a preferred embodiment, the method for producing liposomes used in the present invention can be carried out by performing the step c) and the step b) in order after the step a).

  In one embodiment, in the method for producing liposomes used in the present invention, step c) comprises (c1) a crosslinking agent (for example, 3,3′-dithiobis (sulfosuccinimidyl propionate)). A step of adding a solution A containing a carbonate buffer solution to the powder A containing the solution to dissolve it to prepare a mixed solution; and (c2) adding the mixed solution to a solution containing the liposomes at room temperature for 16 to 20 Stir for a period of time, ultrafilter at a molecular weight cut off of 30,000, and desalinate to prepare a solution containing fluorescently encapsulated liposomes bound with 3,3′-dithiobis (sulfosuccinimidyl propionate). And b) the step of (b1) concentrating the solution containing the fluorescently encapsulated liposome and adding the solution A to the concentrated solution containing the fluorescently encapsulated liposome; and (b2) ) The solution A is added The mixed solution containing the fluorescently encapsulated liposomes is ultrafiltrated by centrifugation at a molecular weight cut off of 300,000, concentrated, and the solution A is added to the concentrated mixed solution to make it hydrophilic. A step of preparing the fluorescently encapsulated liposome, wherein the step d) comprises (d1) completely dissolving a desired sugar chain or antibody in purified water, and a concentration of 1 to 10 mM (preferably a concentration of 5 mM). (D2) If necessary, ammonium bicarbonate (pH 7-14) is added to the aqueous sugar chain / antibody solution at a concentration of about 0.2 to 1.0 g / mL (preferably Is added at a concentration of 0.6 g / mL) and stirred at 20 to 40 ° C. (preferably 37 ° C.) for 3 to 7 days (preferably 3 days) and refrigerated (for example, about 2 to 8 ° C.) Under, ink for 20 to 60 minutes (preferably 30 minutes) (B) preparing an aminated sugar chain or antibody solution by filtering with a filtration filter; (d3) adding the aminated sugar chain or antibody solution to the solution containing the hydrophilized fluorescently encapsulated liposome; And mixing, and then reacting at room temperature (for example, in the range of about 15 to 30 ° C., preferably in the range of 20 to 25 ° C.) for 2 to 6 hours (preferably 2 hours); and (d4 ) To the reaction solution obtained by the step (d3), a solution B containing a Tris buffer is added, and the mixture is added at room temperature (for example, in the range of about 15 to 30 ° C, preferably 20 to 25 ° C) at The mixture is stirred for 6 hours (preferably 2 hours), and further stirred for 16 to 48 hours (preferably 16 to 20 hours) under refrigeration (for example, 1 to 10 ° C., preferably 2 to 8 ° C.). Ultrafiltration with molecular weight cut off 30,000 A step of producing a sugar chain or antibody-modified liposome containing the fluorescent dye, wherein the step e) concentrates the solution containing the sugar chain or antibody-modified liposome containing the fluorescent dye (e1). , 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) buffer solution C is added, ultrafiltered with a molecular weight cut off of 30,000, concentrated, and concentrated. A step of adding the solution C to a solution containing a sugar chain or an antibody-modified liposome containing a fluorescent dye so as to make the fluorescently encapsulated liposome bound with the sugar chain or antibody hydrophilic may be included. When the primary amino group already exists in the sugar chain or the antibody, the step of preparing the aminated sugar chain solution of (d2) can be omitted. When carrying out the amination reaction, the buffer for dissolving the sugar chain or the antibody may be any buffer as long as it does not have a primary amino group. “Refrigeration” is because the dissolution of a sugar chain or antibody in a buffer containing a primary amino group hinders binding to liposomes means a temperature in the range of about 1-12 ° C., preferably about 2-8 ° C.

  The Tris buffer used in this method is a buffer solution using trishydroxymethylammonium (Tris-hydroxymethylammonium) as a base component, such as N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer. Can be used.

  Examples of sugar chains that can be used in the present method include, but are not limited to, sialyl Lewis X, α1-6 mannobiose, and the like. The sugar chain may preferably be sialyl Lewis X.

  In another embodiment, the sugar chain that can be used by the method of the present invention can be a sugar chain capable of glycosylation amination reaction.

  The amount of sugar chain added can range from about 1 to 250 μL per mL of liposome solution. Preferably, it may be about 2.5-125 μL per mL of liposome solution. When a sugar chain specific to the inflammatory system is used, the sugar chain can be preferably added at a final concentration of 5 mM.

  As used herein, “refrigerated (lower)” refers to a temperature in the range of about 1 ° C. to about 12 ° C., preferably about 2 ° C. to about 8 ° C. As used herein, “room temperature” refers to a temperature in the range of about 15 ° C. to about 30 ° C., preferably about 20 ° C. to about 25 ° C.

(Sugar chain synthesis)
The sugar chain that can be used in the sugar chain-modified liposome used in the present invention can be synthesized by a general sugar chain synthesis method. These methods include (1) chemical synthesis, (2) fermentation using genetically modified cells or microorganisms, (3) synthesis using sugar hydrolase (glycosidase), (4) glycosyltransferase And a method of synthesis using (glycosyltransferase). (WO 2002/081723, JP-A-9-31095, JP-A-11-42096, JP-A-2004-180676, Kenichi Hatanaka, Shinichiro Nishimura, Goro Ouchi and Kazuyoshi Kobayashi (1997) Glucose Science and Industry, Kodansha See Tokyo etc.).

  In the present invention, the sugar chain used in the sugar chain-modified liposome may be a sugar chain synthesized by the above method or a commercially available sugar chain.

(Linkage of sugar chains to liposomes)
In the present invention, any of the above sugar chains may be directly bonded to the liposome prepared as described above, and further, a sugar chain may be bonded via a linker. At this time, the type of sugar chain to be bound to the liposome is not limited to one, and a plurality of sugar chains may be bound. In this case, the plurality of sugar chains may be a plurality of sugar chains having binding activity for different lectins that are commonly present on the cell surface of the same tissue or organ, or may be present on the cell surface of different tissues or organs. It may be a sugar chain having binding activity for different lectins. By selecting a plurality of sugar chains such as the former, it is possible to reliably direct a specific target tissue or organ, and by selecting a plurality of sugar chains such as the latter, a single type of liposome has a plurality of sugar chains. Targets can be directed, and multipurpose target-directed liposomes can be obtained.

  In order to bind the sugar chain to the liposome, it is possible to mix the linker and / or sugar chain at the time of manufacturing the liposome and to bond the sugar chain to the surface while manufacturing the liposome. It is desirable to prepare a glycan and a sugar chain separately, and bind a linker and / or a glycan to a liposome that has been manufactured. This is because the density of the sugar chain to be bound can be controlled by binding the linker and / or sugar chain to the liposome. Direct binding of sugar chains to liposomes can be performed by the method described below.

  A liposome is produced by mixing a sugar chain as a glycolipid, or the sugar chain is bound to the phospholipid of the liposome after production and the sugar chain density is controlled. When the sugar chain is bound using a linker, it is preferable to use a biological protein, particularly a human-derived protein, as the linker. The protein derived from the living body is not limited, and examples thereof include proteins existing in blood such as albumin and other physiologically active substances existing in the living body. Examples include animal serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA). Particularly when human serum albumin is used, it has been confirmed by experiments on mice that the uptake into each tissue is large. It has been. The liposome used in the present invention is very stable, and can be post-treated by binding a protein, binding a linker, or binding a sugar chain after forming the liposome. Therefore, it is possible to produce various liposomes according to the purpose by producing a large amount of liposomes and then binding different proteins according to the purpose or by binding a linker or sugar chain.

  In the liposome used in the present invention, a sugar chain is directly bonded to a lipid constituting the liposome via a linker. This liposome has a complex carbohydrate type ligand such as glycolipid and glycoprotein and is hydrophilized with a low molecular weight compound.

  In the pharmaceutical composition of the present invention, the hydrophilized liposome contained therein may contain a compound having a pharmaceutical effect. The compound having a pharmaceutical effect may be encapsulated in the liposome or bound to the surface of the liposome, but a protein having a pharmaceutical effect may be used as a linker. In this case, the protein may also serve as a linker for binding the liposome and sugar chain and a protein having a medicinal effect. Examples of proteins having medicinal effects include physiologically active proteins.

  In the production of the sugar chain or antibody-modified liposome used in the present invention, a crosslinking group may be used when binding the liposome and the linker.

  In order to bind a sugar chain or an antibody to a liposome via a linker, the method described below may be used.

First, proteins are bound to the liposome surface. Liposomes are treated with an oxidizing agent such as NaIO ₄ , Pb (O ₂ CCH ₃ ) ₄ , NaBiO ₃ to oxidize gangliosides present on the membrane surface of the liposome, and then a test drive such as NaBH ₃ CN, NaBH ₄ is used. Then, the linker and the ganglioside on the liposome membrane surface are bound by a reductive amination reaction. This linker is also preferably made hydrophilic, in which a compound having a hydroxy group is bound to Ringer protein. For example, bissulfosuccinimidyl suberate, disuccinimidyl glutarate, dithiobissuccinimid Such as dilpropionate, disuccinimidyl suberate, 3,3′-dithiobis (sulfosuccinimidylpropionate), ethylene glycol bissuccinimidyl succinate, ethylene glycol bissulfosuccinimidyl succinate What is necessary is just to couple | bond the compound used for the above-mentioned hydrophilization, such as a tris (hydroxymethyl) aminomethane, with the linker on a liposome using a bivalent reagent.

  Specifically, first, one end of the divalent reagent for crosslinking is bonded to all amino groups of the linker. Then, glycosylamine compounds obtained by glycosylation amination of various sugar chains or reducing ends of antibodies are prepared, and a part of the above-mentioned cross-linked divalent reagent bonded to the amino group of the sugar chain or antibody and the liposome is prepared. Combine with other unreacted ends. The covalent bond between the sugar chain or antibody and / or hydrophilic compound and the liposome, or the covalent bond between the sugar chain or antibody and / or hydrophilic compound and the linker may be cleaved when the liposome is taken into the cell. Is possible. For example, when a linker and a sugar chain or sugar chain are covalently bonded via a disulfide bond, the sugar chain or antibody is cleaved by reduction in the cell. When the sugar chain or the antibody is cleaved, the liposome surface becomes hydrophobic, binds to the biological membrane, disturbs the membrane stability, and releases the drug contained in the liposome.

  Next, hydrophilicity is obtained by using the unreacted end of most of the divalent reagent remaining unreacted with no sugar chain bound to the surface of the protein on the sugar chain or antibody-modified liposome membrane thus obtained. Process. In other words, the unreacted end of the divalent reagent bound to the protein on the liposome and the compound used for the above-mentioned hydrophilicity such as tris (hydroxymethyl) aminomethane are subjected to a binding reaction to make the entire liposome surface hydrophilic. To do. Hydrophilization of the liposome surface and the linker improves transferability to various tissues, retention in blood, and transferability to various tissues. This is because when the liposome surface and the linker surface are rendered hydrophilic, the parts other than the sugar chains or antibodies appear as if they are in-vivo moisture in each tissue, etc. This is probably due to the fact that only the sugar chain or antibody is recognized by the target tissue (lectin (sugar chain recognition protein) or the like).

  Preferably, the linker is attached to the liposome as follows. (1) Sodium metaperiodate / N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) is added and stirred overnight under refrigeration to oxidize the liposome particle surface. (2) To remove free sodium metaperiodate and exchange the buffer with PBS buffer (pH 8.0), ultrafiltration (fraction molecular weight: 300,000) is performed. (3) To a solution obtained by adding HSA / PBS buffer (pH 8.0) and reacting at room temperature for 2 hours, sodium cyanoborate / PBS buffer (pH 8.0) was added, and the solution was allowed to react at room temperature for 2 hours. Then, stir overnight under refrigeration. (4) Ultrafiltration (fractionated molecular weight: 300,000) is performed for the purpose of removing free sodium cyanoborate and HSA, and exchanging the buffer with a carbonate buffer (pH 8.5). Thereby, a linker can be liposome-bonded.

  Preferably, the linker is attached to the liposome as follows. (1) Sodium metaperiodate / N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) is added and stirred overnight under refrigeration to oxidize the liposome particle surface. (2) To remove free sodium metaperiodate and exchange the buffer with PBS buffer (pH 8.0), ultrafiltration (fraction molecular weight: 300,000) is performed. (3) Add HSA / PBS buffer (pH 8.0) and react at room temperature for 2 hours, and then stir overnight under refrigeration. (4) Perform ultrafiltration (fractional molecular weight: 300,000) for the purpose of removing free HSA and exchanging the buffer with a carbonate buffer (pH 8.5). Thereby, a linker can be liposome-bonded.

  More preferably, the linker is attached to the liposome as follows. (1) Sodium metaperiodate / N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) is added and stirred overnight under refrigeration to oxidize the liposome particle surface. (2) In order to remove free sodium metaperiodate and exchange the buffer with PBS buffer (pH 8.0), the solution was concentrated to 1/5 to 1/10 times by ultrafiltration, and PBS buffer ( Add pH 8.0) to the original volume. This operation is repeated twice. (3) After adding HSA / PBS buffer (pH 8.0) and reacting at room temperature for 2 hours, sodium cyanoborate / PBS buffer (pH 8.0) was added, and after 2 hours at room temperature. Stir overnight under refrigeration. (4) For the purpose of removing free sodium cyanoborate and HSA and exchanging the buffer with carbonate buffer (pH 8.5), the solution is concentrated to 1/5 to 1/10 times by ultrafiltration, and carbonate buffer Add (pH 8.5) to the original volume. This operation is repeated twice. Thereby, a linker can be liposome-bonded.

  More preferably, the linker is attached to the liposome as follows. (1) Sodium metaperiodate / N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) is added and stirred overnight under refrigeration to oxidize the liposome particle surface. (2) In order to remove free sodium metaperiodate and exchange the buffer with PBS buffer (pH 8.0), the solution was concentrated to 1/5 to 1/10 times by ultrafiltration, and PBS buffer ( Add pH 8.0) to the original volume. This operation is repeated twice. (3) Add HSA / PBS buffer (pH 8.0), react at room temperature for 2 hours, and further stir overnight under refrigeration. (4) For the purpose of exchanging the buffer with carbonate buffer (pH 8.5), concentrate to 1/5 to 1/10 times the volume by ultrafiltration, and add carbonate buffer (pH 8.5) to the original volume. . This operation is repeated twice. Thereby, a linker can be liposome-bonded.

The sugar chain or antibody is then bound to a linker on the liposome. For this purpose, a reducing end constituting a sugar chain or an antibody is glycosylated with an ammonium salt such as NH ₄ HCO ₃ or NH ₂ COONH ₄ and then bissulfosuccinimidyl suberate, disuccinimidyl glutarate is used. , Dithiobissuccinimidylpropionate, disuccinimidyl suberate, 3,3′-dithiobis (sulfosuccinimidylpropionate), ethylene glycol bissuccinimidyl succinate, ethylene glycol bissulfosk Using a divalent reagent such as cinimidyl succinate, a linker bound to the liposome membrane surface and the glycosylated amino sugar or sugar chain are bound to obtain a liposome. In addition, those sugar chains or antibodies that are commercially available can be used.

  Preferably, the sugar chain or antibody can be bound to the linker on the liposome as follows. (1) A sugar chain or an antibody is dissolved in purified water and reacted at 37 ° C. for 3 days under ammonium hydrogen carbonate saturation. (Aminated sugar chain or antibody solution). (2) A crosslinking agent DTSSP (PIERCE) is added to the liposome solution, and after 2 hours at room temperature, the mixture is stirred overnight under refrigeration. (3) In order to remove free DTSSP, ultrafiltration (fraction molecular weight: 300,000) is performed. (4) After adding aminated sugar chain or antibody solution and reacting at room temperature for 2 hours, tris (hydroxymethyl) aminomethane / carbonate buffer (pH 8.5) was added and the mixture was kept overnight under refrigeration. Stir. (5) Ultrafiltration (fractionated molecular weight: 300,000) is performed to remove free sugar chains or antibodies and tris (hydroxymethyl) aminomethane, and to exchange the buffer with HEPES buffer (pH 7.2). Thereby, the coupling | bonding to the linker on the liposome of a sugar_chain | carbohydrate or an antibody can be achieved.

  Liposome formation and fluorescence (eg, cy5.5, Cy3, Cy7, etc.) labeled HSA encapsulation (step A); liposome hydrophilization treatment (step B); liposome-HSA binding (step C) and sugar to liposome Finishing can be accomplished by 0.45 μm filter filtration following binding of chain or antibody, etc. (Step D).

  The protein amount of the liposome, sugar chain or antibody-modified liposome used in the present invention can be measured, for example, by the BCA method by measuring the amount of HSA encapsulated in the liposome and the total protein amount of HSA coupled to the liposome surface.

For example, Micro BCA ^™ Protein Assay Reagent kit (catalog number 23235BN) (PIERCE Co. LTD) can be used for the measurement of the amount of protein. As a standard substance, 2 mg / ml albumin (BSA) may be used. (1) As a standard solution, a standard substance (2 mg / ml: albumin) is diluted with a PBS buffer solution to prepare solutions of 0, 0.25, 0.5, 1, 2, 3, 4, 5 μg / 50 μl. (2) Cy5.5-encapsulated sugar chain-modified liposomes are diluted 20-fold with PBS buffer to prepare a sample solution. (3) Dispense 50 μl each of the standard solution and the sample solution into test tubes. (4) Add 100 μl of 3% sodium lauryl sulfate solution (SDS solution) to each test tube. (5) Reagents A, B, and C attached to the kit are mixed so that reagent A: reagent B: reagent C = 48: 2: 50, and 150 μl is added to each test tube. (6) The test tube is allowed to stand at 60 ° C. for 1 hour. (7) After returning to room temperature, the absorbance at 540 nm is measured, a calibration curve is prepared with the standard solution, and the protein amount of the liposome is measured. An example of a calibration curve is shown in FIG.

  The protein amount of the sugar chain-modified liposome used in the present invention may be, for example, in the range of 0.1 to 1 mg / ml. The protein amount of the sugar chain-modified liposome labeled with Cy3 can be, for example, 0.24 mg / ml or more. The protein amount of the sugar chain-modified liposome labeled with Cy5.5 can be, for example, 0.45 mg / ml or more. The protein amount of the sugar chain-modified liposome labeled with Cy7 can be, for example, 0.20 mg / ml or more.

  The amount of the constituent lipid of the liposome or sugar chain-modified liposome used in the present invention can be calculated, for example, by quantifying the amount of cholesterol.

  <Principle of lipid quantification>

  For quantification of lipids, for example, Determiner TC555 kit (catalog number UCC / EAN128) (KYOWA Co. LTD) can be used. As a standard substance, 50 mg / ml cholesterol attached to the kit is used. (1) As a standard solution, a standard substance (50 mg / ml: cholesterol) is diluted with a PBS buffer solution, and 0, 0.1, 0.25, 0.5, 0.75, 1, 5, 10 μg / 20 μl solution To prepare. (2) Cy5.5-encapsulated sugar chain-modified liposomes are diluted 5-fold with a PBS buffer solution to prepare a sample solution. (3) Dispense 20 μl each of the standard solution and the sample solution into test tubes. (4) To each test tube, 17 μl of Triton X-100 (10% solution) is added and stirred, and then allowed to stand at 37 ° C. for 40 minutes. (5) Add 300 μl of the enzyme reagent of Determiner TC555 kit, stir, and then let stand at 37 ° C. for 20 minutes. (6) The absorbance is measured at 540 nm, a calibration curve (an example of a calibration curve is shown in FIG. 5) is prepared from the standard solution, the amount of cholesterol in the liposome is measured, and the amount of lipid is determined.

The conversion formula for obtaining the lipid content from the cholesterol content is expressed, for example, as follows.
Lipid amount (μg / 50 μl) = cholesterol amount (μg / 50 μl) × 4.51 (conversion factor)
The ratio of protein to lipid in the liposome can be derived, for example, from the results of protein quantification and lipid quantification described above. The sugar chain-modified liposome used in the present invention preferably has a protein to lipid ratio of about 0.1 to about 0.5.

  The lipid amount of the sugar chain-modified liposome used in the present invention may be in the range of 1 to 4 mg / ml, for example. The amount of lipid in the sugar chain-modified liposome labeled with Cy3 can be, for example, 1.2 mg / ml or more. The amount of lipid in the sugar chain-modified liposome labeled with Cy5.5 can be, for example, 1.4 mg / ml or more. The amount of lipid in the sugar chain-modified liposome labeled with Cy7 can be, for example, 2.1 mg / ml or more.

  The particle size distribution and particle size of the liposome, sugar chain, or antibody-modified liposome used in the present invention can be determined by, for example, diluting the liposome particle 50 times with purified water to produce a Zetasizer Nano (Nan-ZS: MALVERN Co. LTD). ).

  The liposome, sugar chain or antibody-modified liposome used in the present invention preferably has a particle size of about 50 nm to about 300 nm in the maximum range of the particle size distribution. This is because a particle size of about 50 nm to about 300 nm can avoid recognition of immune system cells such as macrophages and can avoid to some extent uptake from the endothelial reticuloendothelial system (RES) of the liver and spleen. A sugar chain-modified liposome having a particle size of about 50 nm to about 300 nm is suitable for encapsulating a drug and delivering the drug to a target organ or a diseased part.

  The liposome, sugar chain or antibody-modified liposome used in the present invention has an average particle size of about 50 nm to about 300 nm, preferably about 80 nm to about 200 nm, more preferably about 100 nm. This is because if the liposome particle size is too small, it will enter the cell / endothelial system of the liver / spleen non-specifically, and if the particle size is too large, it will be phagocytosed by immune system cells such as macrophages. Moreover, it is desirable that the liposome used in the present invention is negatively charged. By being negatively charged, the interaction with negatively charged cells in the living body can be prevented. The zeta potential of the liposome surface used in the present invention is −120 to 10 mV, preferably −80 to −30 mV, more preferably −50 to −30 mV at 37 ° C. in physiological saline. The zeta potential of the liposome surface can be, but is not limited to, −120 mV to −30 mV at 25 ° C. Preferably, it is less than −30 mV at 25 ° C. The zeta potential on the liposome surface may be less than −120 mV (25 ° C.), or may be −30 mV or more. This is because aggregation between liposomes does not have to occur.

  Examples of drugs that can be included in the liposome, sugar chain or antibody-modified liposome used in the present invention include alkylated anticancer agents, antimetabolites, plant-derived anticancer agents, anticancer antibiotics, BRM / cytokines, platinum complex anticancer agents. , Immunotherapeutic agents, hormonal anticancer agents, tumor drugs such as monoclonal antibodies, central nervous system drugs, peripheral nervous system / sensory organ drugs, respiratory disease drugs, cardiovascular drugs, digestive organ drugs, hormonal drugs Urinary / genital agents, vitamins / nutritional tonics, metabolic drugs, antibiotics / chemotherapeutic drugs, test drugs, anti-inflammatory drugs, eye disease drugs, central nervous system drugs, autoimmune drugs, cardiovascular drugs Lifestyle-related diseases such as diabetes and hyperlipidemia, corticosteroids, immunosuppressants, antibacterial agents, antiviral agents, angiogenesis inhibitors, cytokines, chemokines Gene therapy-related nucleic acid preparations such as anti-cytokine antibodies, anti-chemokine antibodies, anti-cytokine / chemokine receptor antibodies, siRNA, miRNA, smRNA, antisense ODN or DNA, neuroprotective factors, antibody drugs, molecular targeted drugs, osteoporosis / Examples include bone metabolism improving drugs, neuropeptides, bioactive peptides / proteins, and the like. For example, as a tumor drug, alkylation of nitrogen mustard-N-oxide, cyclophosphamide, ifosfamide, pulsphan, nimustine hydrochloride, mitoblonitol, melphalan, dacarbazine, ranimustine, estramustine phosphate sodium, etc. Agents, mercaptopurine, thioinosine (mercaptopurine riboside), methotrexate, enositabine, cytarabine, ancitabine hydrochloride (cyclocytidine hydrochloride), fluorouracil, 5-FU, tegafur, doxyfluridine, carmofur and other antimetabolites, etoposide, bin plastin sulfate , Plant-derived anticancer agents such as pinklistin sulfate, vindesine sulfate, paclitaxel, taxol, irinotecan hydrochloride, nogitecan hydrochloride and other alkaloids, actinomycin Anticancer antibiotics such as mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate, pepromycin sulfate, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride (acracinomycin A), pirarubicin hydrochloride, epirubicin hydrochloride, neocarinostatin In addition, there are mitoxantrone hydrochloride, carboplatin, cisplatin, L-asparaginase, acegraton, procarbazine hydrochloride, tamoxifen citrate, ubenimex, lentinan, schizophyllan, medroxyprogesterone acetate, phosfestol, mepithiostane, epithiostanol and the like. In the present invention, the above-mentioned drugs include derivatives thereof.

  By including the drug as described above, the pharmaceutical composition of the present invention can be used for the treatment of diseases such as arteriosclerosis, cancer and inflammation. Here, cancer includes all neoplastic diseases such as tumors and leukemias. When these drugs are included in the pharmaceutical composition of the present invention and administered, the drugs accumulate at sites of arteriosclerotic lesions, cancer, and inflammation as compared to when the drug is administered alone. Compared to the case of single administration, it can accumulate 2 times or more, preferably 5 times or more, more preferably 10 times or more, and particularly preferably 50 times or more. Compared with the case of administering a liposome (reference liposome) to which tris (hydroxymethyl) aminomethane is bound, it can accumulate 3 to 4 times, preferably 4 to 6 times.

  The pharmaceutical composition of the present invention can be used for treatment of various diseases by encapsulating a drug in a hydrophilic liposome as described above. The pharmaceutical composition can be administered by intravenous injection or by oral administration. In the pharmaceutical composition of the present invention, the medium that has been transferred to the blood by oral administration shows the same tendency as that of intravenous injection when delivered to an organ when orally administered.

  In addition, the compound which has a pharmaceutical effect may be enclosed in a liposome, and may be combined with the liposome surface. For example, proteins can be bound to the surface in the same manner as the above linker binding method, and other compounds can be bound by a known method by utilizing the functional group of the compound. . In addition, the encapsulation inside the liposome is performed by the following method. Well-known methods may be used for encapsulating drugs in liposomes, for example, solutions containing drugs and phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids or long-chain alkyl phosphates, gangliosides, glycolipids Or a drug etc. are enclosed in a liposome by forming a liposome using the lipid containing phosphatidylglycerols and cholesterol.

  Therefore, in the pharmaceutical composition of the present invention, migration to arteriosclerotic lesions, cancer tissues, inflammatory tissues, and various tissues was selectively controlled by encapsulating drugs or genes that can be used for treatment or diagnosis in liposomes. Therefore, it is possible to enhance the efficacy by concentrating therapeutic drugs or diagnostic agents on target cells and tissues, or to reduce side effects by reducing the uptake of drugs to other cells and tissues.

  Further, when the molecular imaging agent of the present invention is used for diagnosis, a labeling compound such as a fluorescent dye or a radioactive compound is bound to the liposome. The labeled compound-bound liposome binds to the affected area, the labeled compound is taken into the affected area cells, and the disease can be detected and diagnosed using the presence of the labeled compound as an index.

(Health and food)
The present invention can also be used in the health / food field. In such a case, the points to be noted when used as an oral medicine as described above should be considered as necessary. In particular, when used as a functional food / health food such as a specific health food, it is preferable to treat it according to a medicine. Preferably, a product obtained by encapsulating or binding a functional food, nutritional supplement or health supplement to the liposome used in the present invention can be used as a food composition. There is no limitation on the functional food, nutritional supplement or health supplement that can be used in the present invention, and any food that has been designed and processed and converted so as to effectively express the food function is included.

  For example, Ginkgo biloba, Echinasia, Saw Palmetto, St. John's Wort, Valerian, Black Cohosh, Milk Thistle, Evening Primrose, Grape Seed Extract, Bil Perry, Feverfew, Tochigi, Soybean, French Coastal Pine, Garlic, Ginseng, Tea, Ginger, Agaricus, Meshimakobu , Purple ipe, active hemicellulose compound (Active-Hemi-Cellulose-Compound: AHCC), yeast beta-glucan, maitake, propolis, brewer's yeast, cereal, plum, chlorella, barley young leaves, green juice, vitamins, collagen, glucosamine, mulberry Leaf, rooibos tea, amino acid, royal jelly, shiitake mycelium extract, spirulina, tanoshita carrot, watercress, plant fermented food, docosahexaenoic acid (DHA), eicosapen Enic acid (EPA), arachidonic acid (ARA), kelp, cabbage, aloe, megsurino wood, hops, oyster meat extract, picdienol, sesame, etc. are exemplified as functional foods, nutritional supplements or health supplements that can be used in the present invention it can. These may be included in the liposome as they are, or a processed product such as an extract may be included. A food composition containing liposomes is taken orally. The liposome to be used may not have a sugar chain bound thereto, and may have a sugar chain that enhances intestinal absorption or a sugar chain targeted to a specific tissue or organ bound thereto. When the composition of the present invention is administered as a food composition, it may be processed into a food such as a liquid beverage, a gel food, or a solid food. Moreover, you may process into a tablet, a granule, etc. The food composition of the present invention can be used as a functional food, a nutritional supplement or a health supplement depending on the type of food contained in the liposome.

  For example, the liposome containing DHA can be used as a functional food, nutritional supplement or health supplement effective for mild senile dementia and memory improvement.

(Liposomes bound with specific factors for cell adhesion molecules)
(Modification of liposome with antibody against cell adhesion molecule)
The present invention can be carried out, for example, by producing a liposome in which a specific factor for a cell adhesion molecule is bound by modifying the liposome with an antibody against a molecule expressed on the arteriosclerotic lesion endothelium. Specifically, antibodies against cell adhesion molecules such as VCAM-1, ICAM-1, E-selectin, and P-selectin, antibodies against extracellular matrix degrading enzymes such as MMP, and the like can be used. In addition, the antibody modification can be performed using a commercially available liposome labeling kit. Commercially available sialyl Lex antigen (SLX) -modified liposomes that are known to specifically bind to E-selectin can also be used.

  In one specific embodiment, Cy3 fluorescently labeled liposomes can be used in an in vitro and ex vivo experimental system, and Cy5.5 fluorescently labeled liposomes can be used in an in vivo experimental system.

(Separation of vascular endothelial cells and adhesion of liposomes to endothelial cells (in vitro))
In another aspect, the present invention also provides a technique for confirming separation of vascular endothelial cells and adhesion of liposomes to endothelial cells. Separation of vascular endothelial cells and adhesion of liposomes to endothelial cells can be confirmed, for example, as follows. The mouse thoracic aorta is removed, treated with collagenase, and then the cells are collected. The cells are confirmed to be endothelial cells by observing cell morphology, staining cells with vascular endothelial cell markers, and taking up acetylated LDL. Lipopolysaccharide is added to the cultured endothelial cells to induce inflammation and induce cell adhesion molecules. Each antibody or SLX modified liposome is added to cultured endothelial cells. The amount of liposome adhered to the endothelial cells is measured by measuring the fluorescence intensity after 24 hours. Consider antibodies or sugar chains that can adhere to endothelial cells most efficiently.

(Verification and in vivo imaging of antibody-modified liposomes in vivo)
When the present invention is used for confirmation of antibody-modified liposomes in vivo and in vivo imaging, an arteriosclerosis disease model mouse is used. Using a fluorescent in vivo imaging apparatus (IVIS) and micro CT, the liposome is intravenously injected from the tail vein of the mouse, and images are taken over time. At the time when the uptake is considered to be the maximum by imaging, the aorta is removed, and the liposomes taken into the sclerotic lesion are confirmed under a fluorescence microscope. By performing both imaging and histological examination, it is possible to select liposomes that are surely incorporated into the sclerotic lesion.

  In order to confirm the fluorescence in the tissue, a fluorescent stereomicroscope equipped with a CCD camera system or the like can be used.

  In the present invention, when antibody modification to a liposome is performed, it can also be carried out by using an antibody labeling kit available from Katayama Chemical Co., Ltd. As an animal imaging apparatus, Xenogen IVIS (registered trademark) Imaging System, micro CT, or the like may be used.

(Liposomes that accumulate in macrophages)
(1) Modification of liposome with antibody against Mφ Using an antibody against a scavenger receptor known to be involved in foaming of Mφ, the liposome is modified by the same method as described above. Specifically, antibodies against LOX-1 (lectin-like oxidized LDL receptor-1), SR-A (class A scavenger receptor), SRBI (class B type I scavenger receptor), CD36, and CD68 can be used.

(2) Mφ collection and liposome phagocytosis (in vitro)
The mouse abdominal cavity is washed with PBS to recover Mφ. Liposomes modified with each antibody or mannose are added to the cultured Mφ. After 24 hours, Mφ phagocytosed liposomes are counted to evaluate phagocytic efficiency. Consider antibodies or sugar chains that are phagocytosed by Mφ most efficiently.

(3) Confirmation of antibody-modified liposome in vivo and in vivo imaging For the atherosclerotic disease model mouse, select a liposome with good phagocytic efficiency in (3) and perform in vivo imaging. The images are taken over time, and the antibodies or sugar chains that are most efficiently taken up in vivo are examined using imaging and histological techniques.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  Hereinafter, although an example explains the composition of the present invention in detail, the present invention is not limited to this. The reagents used in the following were those commercially available from GE Healthcare Bioscience, SIGMA, SAJ, etc. unless otherwise specified.

(Example 1. Preparation of fluorescently encapsulated liposome modified with sialyl Lewis X (SLX))
(Preparation of fluorescently labeled human serum albumin solution)
Cy3 or cy5.5 was used as the fluorescent dye. Human serum albumin / N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) solution (10 mg / ml), (2 ml) was added to cy3 / N-tris (hydroxymethyl) -3-amino. Propanesulfonic acid buffer (pH 8.4) solution (2 mg / ml) or cy5.5 / N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) solution (2 mg / ml), each 1 ml was mixed and stirred at 37 ° C. for 3 hours. This mixed solution was ultrafiltered with a N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) solution with a molecular weight cut-off of 10,000 to remove free fluorescent dyes, and labeled with fluorescence. A labeled human serum albumin solution was prepared.

(Preparation of liposome)
Liposomes were prepared using the improved cholate dialysis method according to a previously reported method (Yamazaki, N., Kodama, M. and Gabis, H.-J. (1994) Methods Enzymol. 242, 56-65). That is, 35 parts by weight of dipalmitoyl phosphatidylcholine, cholesterol, dicetyl phosphate, ganglioside (total ganglioside extract (brain, butammonium salt) distributor: Avanti, catalog number: 10000232 (860053P)) and dipalmitoyl phosphatidylethanolamine, respectively. The mixture was mixed so that the total lipid amount was 45.6 mg at a ratio of 40: 5: 15: 5, 46.9 mg of sodium cholate was added, and the mixture was dissolved in 3 ml of a chloroform / methanol (1: 1) solution. The solution was evaporated and the precipitate was dried in vacuo to obtain a lipid membrane. The obtained lipid membrane was resuspended in 3 ml of TAPS buffered physiological saline (pH 8.4) and stirred at 37 ° C. for 1 hour. The solution was then purged with nitrogen and sonicated to give 3 ml of a clear micelle suspension. To this sonicated micelle suspension, a fluorescently labeled HSA solution completely dissolved to 0.2 mg / 1 ml with HSA buffer (pH 8.4) was slowly added dropwise with stirring and mixed uniformly. The micelle suspension containing this fluorescent dye is subjected to ultrafiltration (fractionated molecular weight: 10,000) using a PM10 membrane (Amicon Co., USA) and TAPS buffered physiological saline (pH 8.4) to obtain a uniform fluorescent dye ( 10 ml of each liposome particle suspension containing cy3 or cy5.5) was prepared.

  The particle size and zeta potential of the liposome particles encapsulating the fluorescent dye in the obtained physiological saline suspension (37 ° C.) are measured by a zeta potential / particle size / molecular weight measuring device (Model Nano ZS, Malvern Instruments Ltd, UK). It was measured. As a result, in the liposome encapsulating cy3, the particle size is about 50 nm to about 300 nm and the zeta potential is −30 to −120 mV, and in the liposome encapsulating cy5.5, the particle size is about 50 nm to about 300 nm, the zeta potential. Was -30 to -120 mV.

(Hydrophilic treatment on the surface of liposome lipid membrane encapsulating fluorescent dye)
Each 10 ml of liposome solution encapsulating the fluorescent dye (cy3 or cy5.5) prepared in this example was subjected to ultrafiltration using XM300 membrane (Amicon Co., USA) and carbonate buffer (pH 8.5) ( (Molecular weight cut off: 300,000), and the pH of the solution was 8.5. Next, 10 mg of a crosslinking reagent bis (sulfosuccinimidyl) suberate (BS ³ ; Pierce Co., USA) was added, and the mixture was stirred at room temperature for 2 hours. Thereafter, the mixture was further stirred overnight at 7 ° C. to complete the chemical binding reaction between the lipid dipalmitoylphosphatidylethanolamine and BS ³ on the liposome membrane. Then, this liposome solution was subjected to ultrafiltration (fractionated molecular weight: 300,000) with an XM300 membrane and a carbonate buffer (pH 8.5). Next, 40 mg of tris (hydroxymethyl) aminomethane dissolved in 1 ml of carbonate buffer (pH 8.5) was added to 10 ml of liposome solution. The solution was then stirred at room temperature for 2 hours, then stirred overnight under refrigeration, ultrafiltered with a molecular weight cut off of 300,000 to remove free tris (hydroxymethyl) aminomethane, and the carbonate buffer Was replaced with N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), and the chemical bonding reaction between BS ³ bound to the lipid on the liposome membrane and tris (hydroxymethyl) aminomethane was performed. Completed. As a result, the hydroxyl group of tris (hydroxymethyl) aminomethane was coordinated on the lipid dipalmitoylphosphatidylethanolamine of the liposome membrane to make it hydrated and hydrophilic.

(Binding of human serum albumin (HSA) to the surface of a liposome membrane encapsulating a fluorescent dye)
The binding of human serum albumin (HSA) onto the surface of the liposome membrane was performed according to a previously reported method (Yamazaki, N., Kodama, M. and Gabis, H.-J. (1994) Methods Enzymol. 242, 56-65). The coupling reaction method was used. That is, this reaction was performed by a two-step chemical reaction. First, metaperoxide obtained by dissolving ganglioside present on the surface of each 10 ml of liposome membrane obtained in this example in 1 ml of N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4). 10.8 mg of sodium iodate was added, and the mixture was stirred at 7 ° C. overnight to oxidize periodate. Free filtration of sodium periodate was removed by ultrafiltration (fractional molecular weight: 300,000) with XM300 membrane and PBS buffer (pH 8.0), and N-tris (hydroxymethyl) -3-amino was removed. The propanesulfonic acid buffer was exchanged with PBS buffer (pH 8.0) to obtain 10 ml of oxidized liposomes. To this liposome solution, 20 mg of human serum albumin (HSA) / PBS buffer (pH 8.0) was added and allowed to react at room temperature for 2 hours, and then 100 μl of 2M NaBH ₃ CN / PBS buffer (pH 8.0). The mixture was stirred at room temperature for 2 hours and further overnight under refrigeration, and HSA was bound by a coupling reaction between ganglioside on the liposome and HSA. Then, ultrafiltration (fraction molecular weight: 300,000) was performed to remove free sodium cyanoborate and human serum albumin, and the buffer of this solution was replaced with carbonate buffer (pH 8.5). 10 ml of each bound liposome solution was obtained.

(Preparation of sugar chain)
Sialyl Lewis X (SLX) (manufacturer: CALBIOCHEM, catalog number: 565950) was used as the sugar chain.

  The mass of the sugar chain was measured and pretreated for use in the following.

(Linkage of sugar chain onto liposome membrane surface-bound human serum albumin (HSA) encapsulating fluorescent dye and hydrophilic treatment of linker protein (HSA))
2 mg of the sugar chain prepared in this example was dissolved in purified water, added to a 0.5 ml aqueous solution in which 0.25 g of NH ₄ HCO ₃ was dissolved, stirred at 37 ° C. for 3 days, and then filtered through a 0.45 μm filter. Thus, the amination reaction of the reducing end of the sugar chain was completed to obtain 4 mg / ml of the glycosylamine compound of the sugar chain (aminated sugar chain solution). Next, 10 mg of a crosslinking reagent 3,3′-dithiobis (sulfosuccinimidylpropionate) (DTSSP; Pierce Co., USA) was added to 10 ml of a part of the liposome solution obtained in this example, and the mixture was stirred for 2 hours at room temperature. Subsequently, the mixture was stirred overnight under refrigeration and ultrafiltered (fractionated molecular weight: 300,000) with an XM300 membrane and carbonate buffer (pH 8.5) to remove free DTSSP. 10 ml of liposomes bound to HSA. Next, the above glycosylamine compound (aminated sugar chain solution) 12.5, 37.5, 125, 250, 500, 1250, 2500 μl is added to this liposome solution, and the mixture is reacted at room temperature for 2 hours. Methyl) aminomethane / carbonate buffer (pH 8.5) was added and then stirred overnight under refrigeration to allow the glycosylated amine compound to bind to DTSSP on liposome membrane bound human serum albumin. Free sugar chains and tris (hydroxymethyl) aminomethane were removed by ultrafiltration (fraction molecular weight: 300,000) with XM300 membrane and HEPES buffer (pH 7.2). As a result, 10 ml of each liposome in which sugar chains, human serum albumin and liposomes were bound was obtained. When a sugar chain-modified liposome was prepared with a modified binding density of 0.025 mg sugar chain / mg lipid, in cy3, the total lipid amount was 43.6 mg, the total protein amount was 4.1 mg, the average particle size was 85.3 nm, and cy5.5, The total lipid amount was 38.5 mg, the total protein amount was 5.8 mg, and the average particle size was 98.3 nm.

  The particle size and zeta potential of the liposome particles encapsulating the fluorescent dye in the obtained physiological saline suspension (37 ° C.) are measured by a zeta potential / particle size / molecular weight measuring device (Model Nano ZS, Malvern Instruments Ltd, UK). It was measured. As a result, the sugar chain-modified liposome has a particle size of about 50 nm to about 300 nm and a zeta potential of −30 to −120 mV for cy3, and a particle size of about 50 nm to about 300 nm and a zeta potential of cy5.5. It was -30 to -120 mV.

(Preparation Example 1. Preparation of fluorescently encapsulated liposome with no sugar chain attached)
(Binding of tris (hydroxymethyl) aminomethane on liposomal membrane-bound human serum albumin (HSA))
In order to prepare a fluorescently encapsulated liposome without a sugar chain as a comparative sample, the liposome obtained in Example 1 (binding of human serum albumin (HSA) on the surface of the liposome membrane encapsulating the fluorescent dye) Add 10 mg of the cross-linking reagent 3,3′-dithiobis (sulfosuccinimidylpropionate (DTSSP; Pierce Co., USA)) to a 10 ml portion of the solution and stir at room temperature for 2 hours, followed by refrigeration overnight. Ultrafiltration (fractionation molecular weight: 300,000) with XM300 membrane and carbonate buffer (pH 8.5) was performed to remove free DTSSP, and 10 ml of liposomes in which DTSSP was bound to HSA on the liposomes were obtained. Next, 26.4 mg of tris (hydroxymethyl) aminomethane (Wako Co., Japan) was added to the liposome solution, and the mixture was stirred at room temperature for 2 hours. The mixture was then stirred overnight under refrigeration, and tris (hydroxymethyl) aminomethane was bound to DTSSP on liposome membrane-bound human serum albumin, limited by XM300 membrane and HEPES buffer (pH 7.2). External filtration (fractional molecular weight: 300,000) Hydrophilicity on liposomal membrane-bound human serum albumin (HSA) due to the presence of 26.4 mg of tris (hydroxymethyl) aminomethane already in large excess in this step At the same time, the sample was filtered through a 0.45 μm filter, and the final product, a liposome (as a comparative sample) in which tris (hydroxymethyl) aminomethane subjected to hydrophilic treatment, human serum albumin and liposomes were combined ( Abbreviation: TRIS) 10 ml (in cy3, total lipid amount 43.9 mg, total protein amount 3.7 mg, average particle) 87.7Nm, in cy5.5, total fat content 37.8 mg, total protein 6.5 mg, the average particle diameter 95 nm) was obtained.

  An example of the properties of the SLX-modified liposome prepared in this example is shown in FIG.

(Comparative Example 1. Preparation of fluorescence-encapsulated sugar chain-modified liposomes modified with sugar chains other than SLX)
(1. Sugar chain-modified liposome modified with sugar chain N-acetyllactosamine)
N-acetyllactosamine is used as the sugar chain. N-acetyllactosamine modified liposomes are obtained using the same method as in Example 1.

(Example 2. Accumulation of SLX-added liposome for arteriosclerosis)
In this example, the accumulation property of SLX-added liposomes against arteriosclerosis was examined.

(Production of an arteriosclerosis model)
In this example, six 6-week-old female apolipoprotein E (ApoE) knockout mice (ApoE − / −) 25 mice (Jackson) and 5 LDL-receptor (LDL-R) knockout mice (LDLR − / −) 5 mice (Jackson) Was used.

  These mice were given a high fat diet (Western diet) (Oriental Yeast Industry: F2WTD) for 12 weeks to produce arteriosclerotic mice.

(Effect of administration of SLX-modified liposome)
(Observation of arteriosclerotic lesions at the base of the aorta)
In this example, SLX-modified liposomes were injected into arteriosclerotic mice, and macrophages and vascular endothelial cells in the arteriosclerotic lesion were searched. Specifically, the tissue image of the aortic root was confirmed by HE staining, and macrophages and vascular endothelial cells were detected by the fluorescent antibody method.

  In this example, the apolipoprotein E (ApoE) knockout mouse (ApoE − / −) arteriosclerosis model prepared in Example 1 was used as an atherosclerotic mouse.

  In 15 atherosclerotic mice, 50 μl of SLX-modified liposome prepared in Example 1 was intravenously injected via the tail vein, and the aorta was removed 24 hours later. Specifically, it was lethal by cervical dislocation after anesthesia with diethyl ether. Mice were disinfected with 70% ethanol and then thoracotomy was performed through a median sternotomy. The heart and aorta were exposed while exfoliating surrounding tissues such as fat, and the mass was removed from the base of the aorta to the descending aorta. .

  A tissue specimen was prepared by cutting the aortic base of the aorta extracted into sections of 5 μm thickness.

  A specimen for HE staining was prepared by slicing the aortic root to a thickness of 5 μm using a cryostat, pasting the section on a glass slide and drying it thoroughly, and then subjecting it to staining.

  A specimen for the fluorescent antibody method was prepared by slicing the aortic root to a thickness of 5 μm using a cryostat, pasting the section on a slide glass and drying it thoroughly, and then subjecting it to staining.

(1. HE staining)
The prepared specimen was stained with HE. HE staining was performed as follows.

  The specimen was washed with distilled water and then stained with Mayer's hematoxylin solution for 10 minutes. Thereafter, it was washed with running water for about 5 minutes. Next, after dyeing with eosin solution for 3 minutes, it was washed lightly with distilled water for about 30 seconds. Finally, it was dehydrated with 70% ethanol and 100% ethanol, cleared with xylene, and sealed.

(2. Fluorescent antibody method)
Macrophages and vascular endothelial cells were stained using the fluorescent antibody method.

  The prepared frozen section (5 μm) was washed with PBS and blocked with 5% BSA (bovine serum albumin) at room temperature for 1 hour. Subsequently, this frozen section was reacted with the primary antibody at room temperature for 1 hour 30 minutes. As primary antibodies for staining macrophages, F4 / 80 (rat anti-mouse IgG) (manufacturer: BMA BIOMEDICALS catalog number: T-2008) or MOMA-2 (rat anti-mouse IgG) (manufacturer: serotec catalog number: MCA519G) Was used. As a primary antibody for staining vascular endothelial cells, CD31 (rat anti-mouse IgG) (manufacturer: BD Pharmingen catalog number: 50274) was used. Next, this section was washed with PBS, and a secondary antibody (Alexa Fluor 488 goat anti-rat IgG) (Invitrogen A-21208) was reacted at room temperature for 1 hour. The sections were washed with PBS and stained with Hoechst 33258 (polysciences, Inc. PA, USA catalog 09460). Further, the section was washed with PBS and encapsulated.

  The results are shown in FIGS. FIG. 7 is a HE-stained aortic tissue photograph. From this HE staining, it was confirmed that in the Apolipoprotein knockout mouse loaded with Western diet, a marked atherosclerotic lesion was formed in the aorta. In arteriosclerotic mice administered with SLX-modified liposomes encapsulating Cy3, accumulation of SLX-modified liposomes was observed in the aorta (see FIG. 8 right and FIG. 9 left). In tissue photographs by the fluorescent antibody method, SLX-modified liposomes were stained red, endothelial cells were green, and nuclei were stained blue (left in FIG. 9).

  In the atherosclerotic disease model mouse, it was confirmed that the SLX-modified liposome labeled with fluorescence was taken into the atherosclerotic lesion.

(Comparative Example 2. Accumulation of SLX-modified liposomes in wild type mice)
As controls, 6-week-old female C57BL / 6 mice (Wild-type) (10 mice) were used. The same method as in Example 2 was used except that C57BL / 6 mice were used instead of atherosclerotic mice.

  As a result, in the HE-stained wild-type mouse, no arteriosclerotic lesion was observed in the blood vessel wall. In the fluorescence staining method, in the aorta of wild-type mice, endothelial cells were stained green and nuclei were stained blue, but SLX-modified liposomes encapsulating Cy3 were not detected (right side of FIG. 10).

  For the fluorescently encapsulated liposome without sugar chain prepared in Preparation Example 1 and the N-acetyllactosamine-modified liposome prepared in Comparative Example 1, the same experiment as in Example 2 and this comparative example was conducted, The effects of administering type liposomes and N-acetyllactosamine modified liposomes are evaluated.

(Example 3. Confirmation of atheromatous plaque)
In this example, an aortic sclerosis lesion was observed (ex vivo). Specifically, 50 μl of the SLX-modified liposome prepared in Example 1 was intravenously injected from the tail vein of the mouse, and the aorta was removed 24 hours later. The aorta was developed in the long axis direction, observed with a fluorescence microscope, and it was examined whether liposomes were taken up in plaques. Further, the same specimen was stained with Sudan IV stain.

  Specifically, the LDL-receptor (LDL-R) knockout mouse (LDLR − / −) arteriosclerosis model prepared in Example 2 was used as an animal model.

  The arteriosclerotic mice (3 mice) were anesthetized with diethyl ether, and then the SLX-modified liposome (50 μl) prepared in Example 1 was intravenously injected from the tail vein.

  24 hours after administration of the SLX-modified liposome, the aorta was removed from the mouse using the same method as in Example 2.

  Sudan IV staining was used to search for atheromatous plaques. Specifically, it was performed as follows.

  The excised aorta was fixed overnight with 10% formalin. Subsequently, it was rinsed with 70% ethanol for 3 minutes and stained with 0.5% (wt / vol) Sudan IV staining solution (Sudan IV 1 g, distilled water 30 ml, ethanol 70 ml and acetone 100 ml) at room temperature for 15 minutes. . Stained sections were rinsed with 70% ethanol to decolorize the background. After washing with distilled water for 5 minutes, it was stored in PBS at 4 ° C.

  In addition, the aorta was excised 24 hours later from a mouse intravenously injected with SLX-modified liposomes labeled with Cy3, expanded in the longitudinal direction, and the portion where plaque was observed was observed with a fluorescence microscope. Fluorescence was detected.

  In the LDLR-/-arteriosclerosis model, allotemic plaques were stained with Sudan IV (Figure 12 left). It was confirmed that SLX-modified liposomes were accumulated at this site (FIG. 12 right).

  The use of surface-modified liposomes as a diagnostic method for unstable plaques is a completely new and original method. Fluorescence is accumulated in macrophages by phagocytosing liposomes, so that only arteriosclerotic lesions where highly active macrophages are present can be observed. This makes it possible to identify unstable plaques, and is expected to lead to the development of an innovative diagnostic method for arteriosclerosis with qualitative diagnostic characteristics. When this technology capable of specifically reaching unstable plaque is achieved, it is considered that there is a possibility that it can be used as a new drug delivery system (DDS: drug delivery system) by encapsulating a drug or the like inside the liposome.

  With respect to the liposome with no sugar chain prepared in Preparation Example 1 and the N-acetyllactosamine-modified liposome prepared in Comparative Example 1, the same experiment as in this example was conducted to incorporate the liposome into the atheromatous plaque. evaluate.

(Example 4. Biological imaging of arteriosclerosis model)
In this example, in vivo imaging (in vivo) of an arteriosclerotic lesion of the aorta was performed.

50 μl of the SLX-modified liposome prepared in Example 1 was intravenously injected from the tail vein of the mouse, and 24 hours later, the ROX was set on the chest of the mouse with eXplore Optix ^™ (GE Healthcare) and photographed.

  As the Nembutal solution, a stock solution (pentobarbital sodium 50 mg / ml Dainippon Sumitomo Pharma) diluted 10-fold with physiological saline was used. This Nembutal solution was anesthetized by administering 150 μl into the peritoneal cavity of arteriosclerotic mice. From the tail vein, cy5.5-encapsulated SLX-modified liposome (K1) (50 μl: equivalent to 150 μg of lipid) was administered. Image data were taken with a fluorescence imaging device eXplot Optix (GE Healthcare) at 6 hours and 24 hours after administration. All the image data was taken from the ventral side (FIG. 13 shows the state of photography).

  As a result, it was confirmed that liposomes accumulated in arteriosclerotic lesions at 6 hours and 24 hours after administration of SLX-modified liposomes (FIGS. 1, 2 and 3; FIG. 1 shows 6 hours later). Fig. 2 shows the image after 24 hours, Fig. 3 is an enlarged view of the image after 24 hours (Fig. 2), both are mice with arteriosclerosis on the left side, and wild-type mice on the right side The image is shown.)

(Comparative Example 3. In vivo imaging of wild type mice)
Using the same method as in Example 4, cy5.5-encapsulated SLX-modified liposome (K1) (50 μl: equivalent to 150 μg of lipid) was administered from the tail vein. Image data were taken with a fluorescence imaging apparatus eXplot Optix (GE Healthcare) at 6 hours and 24 hours after administration.

  The same experiment as in Example 4 and this comparative example was conducted for the fluorescence-encapsulated liposome without sugar chain prepared in Preparation Example 1 and the N-acetyllactosamine-modified liposome prepared in Comparative Example 1, and a living body of an arteriosclerosis model was obtained. Perform imaging.

(Summary)
The use of surface-modified liposomes as a diagnostic method for unstable plaques is a completely new and original method. Fluorescence is accumulated in macrophages by phagocytosing liposomes, so that only arteriosclerotic lesions where highly active macrophages are present can be observed. This makes it possible to identify unstable plaques, and is expected to lead to the development of an innovative diagnostic method for arteriosclerosis with qualitative diagnostic characteristics. When this technology capable of specifically reaching unstable plaque is achieved, it may be used as a new drug delivery system (DDS: drug delivery system) by encapsulating a drug or the like inside the liposome. Furthermore, it is believed that the present invention may be able to add a qualitative diagnosis in intima-media thickness (IMT) thickening detected by carotid echo.

(Example 5. Preparation of fluorescently encapsulated liposome modified with antibody against arteriosclerotic lesion)
In this example, instead of SLX as a specific factor for arteriosclerotic lesions, antibodies (antibodies against cell adhesion molecules such as VCAM-1, ICAM-1, E-selectin, P-selectin, and extracellular matrix degradation such as MMP) The same method as in Example 1 is used except that an antibody against an enzyme), an antibody fragment, and an antibody variant are used.

(Preparation of fluorescently labeled human serum albumin solution)
A fluorescently labeled human serum albumin solution is prepared using the same method as in Example 1.

  The fluorescent dye solution was mixed with the human serum albumin / N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer solution and stirred. This mixed solution is ultrafiltered to remove free fluorescent dye, and a fluorescently labeled labeled human serum albumin solution is prepared.

(Preparation of liposome)
Mix the lipids and dissolve in chloroform / methanol solution. The solution is evaporated and the precipitate is dried in vacuo to obtain a lipid membrane. The resulting lipid membrane is resuspended in buffer and stirred. The solution is then purged with nitrogen and sonicated to obtain a micelle suspension. A fluorescently labeled HSA solution is slowly added dropwise to the sonicated micelle suspension while stirring and mixed uniformly, and then subjected to ultrafiltration to obtain a liposome particle suspension containing the fluorescent dye.

  The particle diameter and zeta potential of the liposome particles encapsulating the fluorescent dye in the obtained suspension are measured with a zeta potential / particle diameter / molecular weight measuring device (Model Nano ZS, Malvern Instruments Ltd, UK).

(Hydrophilic treatment on the surface of liposome lipid membrane encapsulating fluorescent dye)
The liposome solution containing the fluorescent dye prepared in this example is subjected to ultrafiltration to adjust the pH. Next, a crosslinking reagent is added and stirred. Then, this liposome solution is subjected to ultrafiltration, and tris (hydroxymethyl) aminomethane dissolved in a buffer solution is added. The solution is then stirred and ultrafiltered to complete the chemical binding reaction between the cross-linking agent bound to the lipid on the liposome membrane and tris (hydroxymethyl) aminomethane. Turn into.

(Binding of human serum albumin (HSA) on the surface of a liposome membrane encapsulating a fluorescent dye)
Sodium metaperiodate dissolved in a buffer solution is added to the liposomes obtained in this example, and the periodate is oxidized by stirring. This solution is subjected to ultrafiltration and the buffer is exchanged to obtain oxidized liposomes. A serum albumin (HSA) solution is added to the liposome solution for reaction, and a NaBH ₃ CN solution is added and stirred. Subsequently, ultrafiltration is performed and the buffer solution is exchanged to obtain an HSA-binding liposome solution.

(Binding of antibody on liposome membrane surface-bound human serum albumin (HSA) encapsulating fluorescent dye and hydrophilic treatment of linker protein (HSA))
Antibody solutions using antibodies against cell adhesion molecules such as VCAM-1, ICAM-1, E-selectin, P-selectin, antibodies against extracellular matrix degrading enzymes such as MMP, antibody fragments thereof, and antibody variants as antibodies To prepare. A cross-linking reagent is added to a part of the liposome liquid obtained in this example, and the mixture is stirred and ultrafiltered to obtain a liposome in which the cross-linking reagent is bound to HSA on the liposome. Next, an antibody solution is added to the liposome solution to cause a reaction, and tris (hydroxymethyl) aminomethane buffer is added and stirred. This solution is ultrafiltered to obtain liposomes in which antibody, serum albumin and liposome are bound.

(Accumulation of antibody-modified liposomes against arteriosclerosis)
Using the same method as in Example 2, the accumulation of arteriosclerosis of liposomes modified with the antibody prepared in this Example is examined. As the arteriosclerosis model, one produced in the same manner as in Example 2 is used.

(Effect of administration of antibody-modified liposome)
Using the same method as in Example 2, the effect of administering the antibody-modified liposome prepared in this Example will be examined. Liposomes modified with antibodies are injected into arteriosclerotic mice to search for macrophages and vascular endothelial cells in arteriosclerotic lesions. Specifically, a tissue image of the aortic root is confirmed by HE staining, and macrophages and vascular endothelial cells are detected by a fluorescent antibody method.

  Experiments are also performed on wild-type mice in the same manner as in Example 2.

(Confirmation of atheromatous plaque)
Using a method similar to that of Example 3, observation of the aortic sclerosis lesion (ex vivo) is performed. Specifically, the liposome modified with the antibody prepared in this example is intravenously injected into the tail vein of the mouse, and then the aorta is removed, expanded in the long axis direction, observed with a fluorescence microscope, and the liposome is taken into the plaque. Examine whether it is. Further, the specimen is stained with Sudan IV, and plaques are observed.

(In vivo imaging of arteriosclerosis model)
In vivo imaging of atherosclerotic lesions in the aorta is performed using the same method as in Example 4. Liposomes modified with the antibody prepared in this example are intravenously injected into the tail vein of mice, and then the ROI is set on the chest of the mouse with eXplore Optix ^™ (GE Healthcare) and imaging is performed. In vivo imaging is similarly performed for wild-type mice.

(Example 6. Preparation of statin-encapsulated liposome modified with specific factor for arteriosclerotic lesion and confirmation of effect of therapeutic agent as DDS)
In this example, the same method as in Example 1 is used except that a statin is used instead of the fluorescent dye. Specific factors for arteriosclerotic lesions include SLX, antibodies against molecules expressed on arteriosclerotic endothelium (antibodies to cell adhesion molecules such as VCAM-1, ICAM-1, E-selectin, P-selectin, MMP, etc. Antibody against extracellular matrix degrading enzymes), antibody fragments, and antibody variants.

(Preparation of statin-encapsulating liposome)
In this example, lovastatin is used as an example. Lovastatin may be synthesized by a known method, or a commercially available product (for example, available from US Merck) can be used.

  Lovastatin-encapsulated liposomes are available from AAPS PharmSciTech. 2007 Mar 23; 8 (1): 24.

  The obtained statin-encapsulated liposome is hydrophilized using the same method as in Example 1. The hydrophilic liposome and linker protein are bound. A sugar chain is bound to the liposome to make it hydrophilic.

(Effect of statin-encapsulated liposomes on arteriosclerosis)
Using the same method as in Example 2, the effect of administering the statin-encapsulating liposome prepared in this Example to a subject is examined. These statin-encapsulating liposomes are injected into arteriosclerotic mice, and the state of arteriosclerotic lesions, macrophages and vascular endothelial cells in the arteriosclerotic lesion are observed. Collect tissue pieces as necessary to confirm the pathology. For example, a tissue image of the aortic root is confirmed by HE staining, and macrophages and vascular endothelial cells are detected by a fluorescent antibody method. The same experiment is performed for wild-type mice.

(Confirmation of atheromatous plaque)
Using a method similar to that of Example 3, observation of the aortic sclerosis lesion (ex vivo) is performed. Specifically, statin-encapsulated liposomes prepared in this example were intravenously injected into the tail vein of mice, and then the aorta was removed, expanded in the longitudinal direction, stained with Sudan IV, plaques were observed, Confirm.

(Judgment of results)
By observing the plaque / arteriosclerotic lesion as described above, the therapeutic effect of the statin-encapsulating liposome can be determined.

(Example 7. Preparation of liposome modified with a specific factor for arteriosclerotic lesion using label other than fluorescent dye)
In this embodiment, as a labeling substance, a contrast agent (iohexol, iopamidol, urografin), an ultrasonic contrast agent (levobist, sonazoid), or a metal complex (gadolinium complex, europium complex, terbium complex) is used instead of a fluorescent dye. Except for this, the same method as in Example 1 is used. Specific factors for arteriosclerotic lesions include SLX, antibodies against molecules expressed on arteriosclerotic endothelium (antibodies to cell adhesion molecules such as VCAM-1, ICAM-1, E-selectin, P-selectin, MMP, etc. Antibody against extracellular matrix degrading enzymes), antibody fragments, and antibody variants.

(Preparation of liposome encapsulating labeling substance)
Using the same method as in Example 1, liposomes encapsulating a labeling substance are prepared.

  The particle diameter and zeta potential of the liposome particles encapsulating the labeling substance in the obtained suspension are measured with a zeta potential / particle diameter / molecular weight measuring apparatus (Model Nano ZS, Malvern Instruments Ltd, UK).

(Hydrophilic treatment on the liposomal lipid membrane surface containing the labeling substance)
The liposome solution containing the labeling substance prepared in this example is subjected to ultrafiltration to adjust the pH. Next, a crosslinking reagent is added and stirred. Then, this liposome solution is subjected to ultrafiltration, and tris (hydroxymethyl) aminomethane dissolved in a buffer solution is added. The solution is then stirred and ultrafiltered to complete the chemical binding reaction between the tris (hydroxymethyl) aminomethane and the cross-linking agent bound to the lipid on the liposome membrane, and the surface of the liposomal lipid membrane is hydrated and hydrophilic. Turn into.

(Binding of human serum albumin (HSA) on the surface of the liposome membrane encapsulating the labeling substance)
Sodium metaperiodate dissolved in a buffer solution is added to the liposomes obtained in this example, and the periodate is oxidized by stirring. This solution is subjected to ultrafiltration and the buffer is exchanged to obtain oxidized liposomes. A serum albumin (HSA) solution is added to the liposome solution for reaction, and a NaBH ₃ CN solution is added and stirred. Subsequently, ultrafiltration is performed and the buffer solution is exchanged to obtain an HSA-binding liposome solution.

(Binding of specific factor to arteriosclerotic lesion on liposomal membrane-bound human serum albumin (HSA) encapsulating labeling substance and hydrophilic treatment of linker protein)
SLX, antibodies (antibodies to cell adhesion molecules such as VCAM-1, ICAM-1, E-selectin, P-selectin, antibodies to extracellular matrix degrading enzymes such as MMP), antibody fragments as specific factors for arteriosclerotic lesions Then, a solution in which the antibody variant is dissolved is prepared. A cross-linking reagent is added to a part of the liposome liquid obtained in this example, and the mixture is stirred and ultrafiltered to obtain a liposome in which the cross-linking reagent is bound to HSA on the liposome. Next, an antibody solution is added to the liposome solution to cause a reaction, and tris (hydroxymethyl) aminomethane buffer is added and stirred. This solution is ultrafiltered to obtain liposomes in which antibody, serum albumin and liposome are bound.

(In vivo imaging of arteriosclerosis model)
In vivo imaging of atherosclerotic lesions in the aorta is performed using the same method as in Example 4. The labeled substance-encapsulated liposome prepared in this example is intravenously injected into the tail vein of a mouse, and then imaging is performed using an MRI apparatus, a computer linked tomography apparatus or the like. In vivo imaging is similarly performed for wild-type mice.

  Based on the results in the mouse, the amount to be used in human diagnosis can be determined. Specifically, the labeled substance-encapsulated liposomes prepared in this example are administered to human arteriosclerosis patients and healthy individuals who have taken informed consent, and the progress is determined by MRI apparatus, computer-linked tomography apparatus, etc. The amount of use and the like can be determined by observing with.

(Example 8. Preparation of labeled substance-encapsulating liposome directed to macrophages)
(Preparation of labeled substance-encapsulated liposome modified with mannose)
In this example, mannose is used as a sugar chain, fluorescent substances, contrast agents (iohexol, iopamidol, urografin), ultrasonic contrast agents (levobist, sonazoid), metal complexes (gadolinium complexes, europium complexes) as labeling substances. The same method as in Example 1 is used except that terbium complex) is used.

(Preparation of labeled human serum albumin solution)
A labeled human serum albumin solution is prepared using the same method as in Example 1.

  The labeling substance solution was mixed with human serum albumin / N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer solution and stirred. This mixed solution is ultrafiltered to remove the free labeling substance, and a labeled human serum albumin solution is prepared.

(Preparation of liposome)
Mix the lipids and dissolve in chloroform / methanol solution. The solution is evaporated and the precipitate is dried in vacuo to obtain a lipid membrane. The resulting lipid membrane is resuspended in buffer and stirred. The solution is then purged with nitrogen and sonicated to obtain a micelle suspension. The labeled HSA solution is slowly added dropwise to the sonicated micelle suspension with stirring and mixed uniformly, and then subjected to ultrafiltration to obtain a liposome particle suspension containing the labeled substance.

  The particle diameter and zeta potential of the liposome particles encapsulating the labeling substance in the obtained suspension are measured with a zeta potential / particle diameter / molecular weight measuring apparatus (Model Nano ZS, Malvern Instruments Ltd, UK).

(Hydrophilic treatment on the liposomal lipid membrane surface containing the labeling substance)
The liposome solution containing the labeling substance prepared in this example is subjected to ultrafiltration to adjust the pH. Next, a crosslinking reagent is added and stirred. Then, this liposome solution is subjected to ultrafiltration, and tris (hydroxymethyl) aminomethane dissolved in a buffer solution is added. The solution is then stirred and ultrafiltered to complete the chemical binding reaction between the cross-linking agent bound to the lipid on the liposome membrane and tris (hydroxymethyl) aminomethane. Turn into.

(Binding of human serum albumin (HSA) on the surface of the liposome membrane encapsulating the labeling substance)
Sodium metaperiodate dissolved in a buffer solution is added to the liposomes obtained in this example, and the periodate is oxidized by stirring. This solution is subjected to ultrafiltration and the buffer is exchanged to obtain oxidized liposomes. A serum albumin (HSA) solution is added to the liposome solution for reaction, and a NaBH ₃ CN solution is added and stirred. Subsequently, ultrafiltration is performed and the buffer solution is exchanged to obtain an HSA-binding liposome solution.

(Binding of mannose on the liposome membrane surface-bound human serum albumin (HSA) encapsulating the labeling substance and hydrophilic treatment of the linker protein (HSA))
A mannose solution is prepared in the same manner as in Example 1. A cross-linking reagent is added to a part of the liposome liquid obtained in this example, and the mixture is stirred and ultrafiltered to obtain a liposome in which the cross-linking reagent is bound to HSA on the liposome. Next, a mannose solution is added to the liposome solution for reaction, and a tris (hydroxymethyl) aminomethane buffer solution is added and stirred. This solution is ultrafiltered to obtain liposomes in which mannose, serum albumin and liposomes are bound.

(Production of labeled substance-encapsulated liposomes modified with antibodies against macrophages)
Liposomes modified with antibodies are prepared in the same manner as in Example 5 except that antibodies, antibody fragments, and antibody variants against a scavenger receptor known to be involved in macrophage foaming are used. Specifically, antibodies against LOX-1 (lectin-like oxidized LDL receptor-1), SR-A (class A scavenger receptor), SRBI (class B type I scavenger receptor), CD36, CD68, antibody fragments thereof An antibody variant is used. The hydrophilization treatment on the liposomal lipid membrane surface, the binding of human serum albumin (HSA) onto the liposomal membrane surface, the binding of sugar chains onto the HSA, and the hydrophilization treatment of the linker protein are the same as in Example 1. To do.

(Accumulation to macrophages)
Using the same method as in Example 2, the accumulation of the liposome prepared in this Example against macrophages and macrophages or diseases, disorders or conditions associated with macrophages is examined.

(Effects of liposome administration)
Using the same method as in Example 2, the effect of administering the liposome prepared in this Example will be examined. Specifically, a labeled substance-encapsulating liposome directed to macrophages is injected into a mouse having a disease, disorder or condition associated with macrophages, and macrophages in the lesion are searched. Specifically, a tissue image is confirmed by HE staining, and macrophages are detected by a fluorescent antibody method.

(Biological imaging)
In vivo imaging of mice with diseases, disorders or conditions associated with macrophages is performed using the same method as in Example 4. The labeling substance-encapsulating liposomes directed to macrophages prepared in this Example are intravenously injected into the tail vein of mice, and then photographed using eXplore Optix ^™ (GE Healthcare), MRI apparatus, computer-linked tomography apparatus, etc. Do it.

(Collecting macrophages and phagocytosis of liposomes by macrophages (in vitro))
The mouse abdominal cavity is washed with PBS to recover Mφ. Liposomes modified with each antibody or mannose are added to the cultured Mφ. After 24 hours, Mφ phagocytosed liposomes are counted to evaluate phagocytic efficiency. Consider antibodies or sugar chains that are phagocytosed by Mφ most efficiently.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  INDUSTRIAL APPLICABILITY The present invention has utility in that it can provide a liposome that can be used as a cell / tissue sensing probe for diagnosing arteriosclerosis, which can be applied in biotechnology, particularly molecular imaging.

  If an unstable plaque containing a large amount of macrophages can be imaged, it is possible to develop a new examination method capable of qualitative diagnosis as well as sclerotic lesion presence diagnosis.

  Fluorescence is accumulated in macrophages by phagocytosing liposomes, so that only arteriosclerotic lesions where highly active macrophages are present can be observed. This makes it possible to identify unstable plaques, and is expected to lead to the development of an innovative diagnostic method for arteriosclerosis with qualitative diagnostic characteristics.

  The present invention can be used as a new drug delivery system (DDS: drug delivery system) by encapsulating a drug or the like inside the liposome by achieving the technology of the present invention that can specifically reach unstable plaque. There is usefulness.

FIG. 1 is a photograph in which SLX-modified liposomes labeled with Cy5.5 were administered from the tail vein to ApoE knockout mice loaded with Western diet, and 6 hours later, Optix in vivo imaging. The left is an ApoE knockout mouse and the right is a wild type. In the ApoE knockout mouse having an arteriosclerotic lesion, a high signal was detected at the lesion site, whereas in the mouse without the arteriosclerotic lesion, no signal was detected. FIG. 2 is a picture of in vivo imaging with Optix 24 hours after administering SLX-modified liposomes labeled with Cy5.5 from the tail vein to ApoE knockout mice loaded with Western diet. The left is an ApoE knockout mouse and the right is a wild type. In the ApoE knockout mouse having an arteriosclerotic lesion, a high signal was detected at the lesion site, whereas in the mouse without the arteriosclerotic lesion, no signal was detected. FIG. 3 shows that 50 μl of Cy5.5-labeled SLX-modified liposomes were administered from the tail vein to ApoE knockout mice with arteriosclerotic lesions and wild-type mice without arteriosclerotic lesions. It is the photograph imaged in vivo. In the ApoE knockout mouse having an arteriosclerotic lesion, a high signal was detected at the lesion site, whereas in the mouse without the arteriosclerotic lesion, no signal was detected. FIG. 4 shows an example of a calibration curve (BCA method) for measuring the protein of the liposome. The X axis is the standard protein concentration (μg / 50 mL), and the Y axis is the absorbance at 540 nm. FIG. 5 shows an example of a calibration curve for measuring the lipid content of liposomes. The X axis is the standard cholesterol concentration (μg / 20 mL), and the Y axis is the absorbance at 540 nm. 6 shows the particle size distribution of the liposomes used in the present invention (dynamic scattering method; X-axis is the liposome diameter (nm) and Y-axis is the intensity (%). It is a distribution map immediately after the production, and the lower distribution diagram is a distribution diagram after 6 months after the production, and it is shown that it exists stably even after 6 months after the production.), And its electron microscope (TEM) photograph An example is shown. FIG. 7 is a photograph of a section (5 μm) at the base of the aorta of an ApoE knockout mouse loaded with Western diet and observed with an optical microscope. Significant plaque formation is observed on the vessel wall. FIG. 8 is a photograph of ApoE knockout mice loaded with Western diet administered SLX-modified liposomes labeled with Cy3 from the tail vein, the aorta was taken out 24 hours later, and the aortic root was sectioned (5 μm) and observed. . The left is a photograph of HE staining, and the right is the same site observed with a fluorescence microscope. A red signal of Cy3 was recognized in the plaque, and it was confirmed that the liposome was incorporated in the plaque. FIG. 9 shows that ApoE knockout mice loaded with Western diet were administered SL3 modified liposomes labeled with Cy3 from the tail vein, 24 hours later, the aorta was taken out, the aortic root was sectioned (5 μm), and the vascular endothelium It is the photograph which carried out the fluorescence dyeing | staining of the cell using CD31 as a marker. The left is a photograph of HE staining, and the right is a photograph observed with a fluorescence microscope. Green is vascular endothelial cell, red is liposome, blue is nucleus. A red signal of Cy3 was observed inside the plaque under the vascular endothelial cell, and it was confirmed that the liposome was taken up in the plaque. FIG. 10 shows that ApoE knockout mice loaded with Western diet were administered SLX-modified liposomes labeled with Cy3 from the tail vein, 24 hours later, the aorta was taken out, the aortic root was sectioned (5 μm), and macrophages were further removed. It is the photograph which carried out fluorescence dyeing using F4 / 80 as a marker. The left is a photograph of HE staining, and the right is a photograph observed with a fluorescence microscope. Green is macrophage, red is liposome, blue is nucleus. A red signal of Cy3 was observed inside the plaque, and it was confirmed that a large number of macrophages were present at the same site as liposomes were taken into the plaque. Fig. 11 shows that SLX-modified liposomes labeled with Cy3 were administered from a tail vein to a control mouse (wild-type mouse), 24 hours later, the aorta was removed, the aortic root was sectioned (5 µm), It is the photograph which stained the endothelial cell using CD31 as a marker. The left is a photograph of HE staining, and the right is a photograph observed with a fluorescence microscope. Green is vascular endothelial cell, red is liposome, blue is nucleus. In control mice, no arteriosclerotic lesions were observed (that is, normal blood vessels), and no red Cy3 signal was observed in the blood vessel wall, confirming that liposomes were not taken up into normal blood vessels. FIG. 12 shows that LDLR knockout mice loaded with Western diet were administered SLX-modified liposomes labeled with Cy3 from the tail vein, 24 hours later, the aorta was removed, the aorta was expanded in the longitudinal direction, and Sudan IV was used. It is a dyed photograph. The left is a picture of Sudan IV. The portion where the plaque was formed was stained red. On the right is a photograph of the plaque portion observed with a fluorescence microscope. A red Cy3 signal was detected only from the plaque portion, and it was confirmed that the liposome was incorporated only in the plaque portion. FIG. 13 is a photograph showing the state of the experiment using the fluorescence imaging apparatus.

Claims (28)

A pharmaceutical composition for diagnosing, treating or preventing an arteriosclerotic lesion, comprising a hydrophilic liposome and a specific factor for arteriosclerotic lesions. The pharmaceutical composition according to claim 1, wherein the specific factor for atherosclerotic lesion is selected from the group consisting of sialyl Lewis X (SLX), SLX group, mannose, mannose group, antibody, antibody fragment and antibody variant. The pharmaceutical composition according to claim 1, wherein the specific factor for atherosclerotic lesion is sialyl Lewis X (SLX) or SLX group. The pharmaceutical composition according to claim 1, wherein the diagnosis is a diagnosis of unstable plaque. The pharmaceutical composition according to claim 1, wherein the hydrophilization is by a tris (hydroxymethyl) methylamino group. A part of the lipid constituting the liposome is bonded to a hydrophilic compound cross-linking group-W-tris (hydroxymethyl) methylamino group and NH-C (= O), where W is C (= The pharmaceutical composition according to claim 5, which is an O) -NH bond. The pharmaceutical composition according to claim 2, wherein the liposome and the SLX group are bound via a human serum albumin group. A part of the lipid constituting the liposome is bonded to human serum albumin group-A-linker protein cross-linking group-X-SLX group and CH ₂ —NH, where A is NH—C (═O The pharmaceutical composition according to claim 7, wherein X is a C (═O) —NH bond. The pharmaceutical composition according to claim 2, wherein the SLX group is present on the surface of the liposome. A diagnostic composition for diagnosing arteriosclerotic lesions, comprising:
A) a hydrophilized liposome;
B) specific factors for arteriosclerotic lesions;
C) A diagnostic composition comprising a labeling substance. The diagnostic composition according to claim 10, wherein the specific factor for atherosclerotic lesion is selected from the group consisting of sialyl Lewis X (SLX), SLX group, mannose, mannose group, antibody, antibody fragment and antibody variant. . The diagnostic composition according to claim 11, wherein the specific factor for the atherosclerotic lesion is sialyl Lewis X (SLX) or an SLX group. 11. A diagnostic composition according to claim 10 for use in magnetic resonance imaging. The diagnostic composition according to claim 10, wherein the diagnosis is a diagnosis of unstable plaque. The diagnostic composition according to claim 10, wherein the hydrophilization is based on a tris (hydroxymethyl) methylamino group. A part of the lipid constituting the liposome is bonded to a hydrophilic compound cross-linking group-W-tris (hydroxymethyl) methylamino group and NH-C (= O), where W is C (= The diagnostic composition according to claim 15, which is an O) -NH bond. The diagnostic composition according to claim 11, wherein the liposome and the SLX group are bound via a human serum albumin group. A part of the lipid constituting the liposome is bonded to human serum albumin group-A-linker protein cross-linking group-X-SLX group and CH ₂ —NH, where A is NH—C (═O The diagnostic composition according to claim 17, wherein X is a C (═O) —NH bond. The diagnostic composition according to claim 11, wherein the SLX group is present on the surface of the liposome. The diagnostic composition according to claim 10 for diagnosing an arteriosclerotic lesion by magnetic resonance imaging, wherein the labeling substance is a substance that can be detected by nuclear magnetic resonance imaging. . The diagnostic composition according to claim 20, wherein the labeling substance is a paramagnetic metal. A method for treating or preventing an arteriosclerotic lesion comprising the step of administering an effective amount of the pharmaceutical composition according to claim 1 to a patient in need of treating or preventing an arteriosclerotic lesion. The method of inclusion. A method for detecting an arteriosclerotic lesion, the method comprising:
Administering an effective amount of the diagnostic composition according to claim 10 to a patient in need of detecting an arteriosclerotic lesion; and, in the patient, labeling substance contained in the diagnostic composition with magnetic resonance imaging. And the presence of the labeling substance comprises the step of indicating that the patient has an arteriosclerotic lesion. Use of a compound containing a specific factor for arteriosclerotic lesions in the manufacture of a medicament for treating, preventing, detecting or diagnosing atherosclerotic lesions. A disease, disorder or condition associated with macrophages or macrophages comprising hydrophilic liposomes and specific factors for macrophages selected from the group consisting of mannose, mannose groups, antibodies, antibody fragments and antibody variants, A pharmaceutical composition for diagnosis, treatment or prevention. 26. The pharmaceutical composition according to claim 25, wherein the diagnosis, treatment or prevention comprises targeting only arteriosclerotic lesions in which highly active macrophages are present. A diagnostic composition for diagnosing a macrophage or a disease, disorder or condition associated with macrophages, comprising:
A) a hydrophilized liposome;
B) a specific factor for macrophages selected from the group consisting of mannose, mannose groups, antibodies, antibody fragments and antibody variants;
C) A diagnostic composition comprising a labeling substance. The diagnostic composition according to claim 27, wherein the diagnosis includes diagnosing only an arteriosclerotic lesion in which highly active macrophages are present.